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The Structure and Expression of

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THE STRUCTURE AND EXPRESSION OF THE GENE FOR MOSQUITO
VITELLOGENIC CARBOXYPEPTIDASE

By
Kirk w. Deitsch

A DISSERTATION

Submitted to
Michigan Stage University
in partial fulfillment of the requirements
for the degree of

DOCTOR OF PHILOSOPHY
Graduate Program in Genetics

1994

ABSTRACT

THE STRUCTURE AND EXPRESSION OF THE GENE FOR MOSQUITO
VITELLOGENIC CARBOXYPEPTIDASE

BY

Kirk W. Deitsch

At the heart of the reproductive cycle of the blood-feeding mosquito,
Aedes aegypti, is vitellogenesis, the production of yolk proteins by the fat body
and their subsequent storage in the developing eggs. The genes involved in
yolk protein synthesis are regulated by a complicated system of control that
ultimately leads to their sex-, stage- and tissue-specific expression. Several
hormones have been implicated in this control, but the exact nature of their
action remains unclear. To understand this system further, a small 53kDa yolk
protein was chosen as the object of a detailed investigation into the regulation of
its production. A putative cDNA was cloned for this protein and verified through
direct N-terminal sequencing of the purified protein. This cDNA hybridized to a
1.5 kb mRNA present only in the fat bodies of vitellogenic females. The
deduced amino acid sequence and experiments using the serine protease
inhibitor, [3H] diiisopropyl fluorophosphate, indicated that this protein was a
serine carboxypeptidase. This finding that an enzyme is produced by an
extraovarian tissue and accumulated by oocytes for use in embryonic
development represents a biological phenomenon not previously known for
oviparous animals. The protein was therefore name vitellogenic
carboxypeptidase (VCP).

Two alleles for the gene encoding VCP were cloned from an Aedes

aegypti genomic library, sequenced and analyzed. Several putative regulatory

elements were found, including a 16 bp repeated imperfect palindrome, a TATA
box, a region resembling the fat body enhancer found in Drosophila, and an
arthropod transcriptional initiator. Primer extension analysis showed a single
start site within this initiator. The palindromic element found repeated upstream
of the gene resembled several defined steroid hormone response elements.
The gene for VCP can be induced by treatment with the steroid hormone 20-
hydroxyecdysone (20E), however, this putative response element did not meet
the consensus thought to be necessary for binding of the ZOE/receptor complex,
leading to the hypothesis that the control of 20E on yolk protein synthesis in
mosquito may be indirect. Experiments done with the protein synthesis inhibitor
cycloheximide confirmed this hypothesis. A 20E response cascade therefore
probably exists controlling the expression of the genes involved in mosquito
vitellogenesis. Analysis of the structure and expression of the gene for VCP
represents a first step in the illucidation of this important hormone response

hierarchy.

AKNOWLEDGMENTS

Special thanks to my professor, Dr. Alexander Raikhel, for his advice and
guidance throughout the course of this study and to my committee, Dr. Thomas
Freidman, Dr. Zachary Burton and Dr. Jerry Dodgson, for their help in meeting
the requirements for my degree. In addition, thanks to Wen-Long Cho and
Tolarchan Dhadialla for their assistance in the early stages of this project. All of
my colleagues in the lab, Marianna Kapitskaya, Vladimir Kokoza, Tom
Sappington, Alan Hays, Katya Snigerevskaya, Neal Dittmer, and Jeng-Shong
Chen, as well as Dr. Suzanne Theim and Chi-Zu Chen have had a hand in this
work through discussions and lab talks, and have been extremely helpful.

I would also like to thank Jeng-Shong Chen and Neal Dittmer for their
friendship and camaraderie. They helped make the last four and one half years
an enjoyable experience both inside and outside the lab. Their friendship will
always be valued.

Particular acknowledgment is owed to my parents, William and Connie
Deitsch. All that I achieve in life is ultimately a credit to their patience, guidance,
encouragement, and steadfast loyalty. They have always been there to provide
me with the advice and assistance needed to help me overcome whatever
obstacles I encountered. I only hope to be as good a parent to their
grandchildren as they were to me.

Lastly, I must thank Kelly Hallman; my wife, confidant, counselor,
playmate, best friend and ultimate source of sanity and inspiration. She
provided me with the motivation and desire needed at times when things did not
go well, and helped me enjoy the successes that came along the way. Marrying

her has been my greatest achievement.
iv

CONTENTS

Page
LIST OF TABLES ......................................................................................... vii
LIST OF FIGURES ....................................................................................... viii
ABBREVIATIONS ......................................................................................... x
CHAPTER 1: Introduction .................................................................. 1
References ................................................................... 18
CHAPTER 2: An Extraovarian Protein Accumulated in
Mosquito Oocytes is a Carboxypeptidase
Activated in Embryos ................................................... 22
Abstract ........................................................................ 23
Introduction .................................................................. 24
Materials and Methods ................................................. 25
Results and Discussion ................................................ 28
References ................................................................... 45
CHAPTER 3: Cloning and Analysis of the Locus for
Mosquito Vitellogenic Carboxypeptidase ..................... 48
Abstract ........................................................................ 49
Introduction .................................................................. 50
Results ......................................................................... 53
Discussion .................................................................... 70
Experimental Procedures ............................................. 73
References ................................................................... 77

V

Page

CHAPTER 4: Indirect Control of Yolk Protein Genes
by 20—hydroxyecdysone in the Fat Body
of the Mosquito, Aedes aegypfi ................................... 80
Abstract ........................................................................ 81
Introduction .................................................................. 82
Materials and Methods ................................................. 84
Results ......................................................................... 87
IDEcusflon .................................................................... 104
References ................................................................... 108

CHAPTER 5: Summary and Future Research Prospects... ............... 111
Summary ...................................................................... 1 12
Future Research Prospects ......................................... 115
References ................................................................... 127

vi

LIST OF TABLES

Page
Table 1-1 A sample of the costs of recent epidemics of
mosquito-bome diseases in several regions
of the world ............................................................................. 4
Table 2-1 Homology of mosquito VCP to serine .
carboxypeptidases ................................................................ 37

vii

CHAPTER 1

Figure 1

Figure 2

CHAPTER 2

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

CHAPTER 3

Figure 1
Figure 2
Figure 3

Figure 4

Figure 5

Figure 6

LIST OF FIGURES

Page
Structure of the two major hormones controlling
mosquito vitellogenesis ........................................................... 9
Summary of events in the fat body during the
first cycle of egg production in the bloodfeeding
mosquito, Aedes aegypti ......................................................... 12
Northern analysis of sex- and stage-specific
expression of the VCP mRNA transcript ................................. 30
Northern blot analysis of tissue-specific expression
of the VCP mRNA transcript ................................................... 32
Nucleotide and deduced amino acid sequences
of mosquito VCP ..................................................................... 35
Alignment of amino acid sequences of mosquito
VCP and WCP ........................................................................ 4O
Immunoblot and [3H] OF P binding analyses of VCP
during mosquito development ................................................. 42
Restriction maps of the two alleles of VCP ............................. 55
Genomic blots of DNA from individual mosquitoes ................. 57
Complete genomic sequence of VCP1 ................................... 60
Identification of the transcription start site for VCP
as well as a putative arthropod initiator .................................. 64
Response element comparisons ............................................. 66
Fat body enhancer comparisons ............................................. 69

viii

CHAPTER 4
Figure 1 Effects of 20E concentrations on V9 and total

protein production in cultured fat bodies ................................. 89
Figure 2 Effect of 20E concentrations on V9, VCP and actin

mRNA production in cultured fat bodies ................................. 91
Figure 3 Inhibition of V9 and total protein synthesis by

cycloheximide ......................................................................... 94
Figure 4 Effect of Chx on V9, VCP and actin mRNA production ........... 96
Figure 5 Recovery of vitellogenesis after removal of Chx ..................... 100
Figure 6 Recovery of V9 and VCP mRNA production after

removal of Chx ........................................................................ 103
CHAPTER 5
Figure 1 Southern blots showing the repetitive nature of the

DNA in the region downstream of the VCP locus .................. 118

Figure 2 Diagram of the construct to be used in lipofection
experiments ............................................................................ 122

ABBREVIATIONS

Alnr ......................................................................................... arthropod initiator
ATF ....................................................................... activating transcription factor
BBF-2 ..................................................... Drosophila Box B-binding factor-2
bp ...................................................................................................... base pair
cAMP ................................................................... cyclic adenine monophosphate
cDNA .................................................................................... complementary DNA
Chx .............................................................................................. cycloheximide
CREB .................................................................. CAMP responsive transcriptional
enhancer binding protein

DFP ......................................................................... diisopropyl fluorophosphate
DNA .................................................................................. deoxyribonucleic acid
DSX ...................................................................... Drosophila double sex protein
20E .................................................................................... 20-hydroxyecdysone
EcR ....................................................................................... ecdysone receptor
EcRE ........................................................................ ecdysone response element
EDTA ................................................................ ethylenedinitrilo tetraacetic acid
606 .............................................. Compusoftware from University of Wisconsin

Genetics Computer Group

JHllI ..................................................................................... juvenile hormone Ill
kDa ..................................................................................................... kiIoDalton
kb ............................................................................................... kilobase pairs

X

M, .......................................................................................... molecular weight

mRNA ................................................................................ messenger RNA
PBM ............................................................................................ post blood meal
PCR ........................................................................... polymerase chain reaction

RNA ........................................................................................... ribonucleic acid
808 ................................................................................ sodium dodecyl sulfate
SDSIPAGE ........................................... SDS polyacrylamide gel electrophoresis

TCA ...................................................................................... trichloroacetic acid
UGAL ......................................... University of Georgia at Athens laboratory strain
VCP ...................................................................... vitellogenic carboxypeptidase

VCP‘I ......................................................................... first allele of the VCP gene
VCP2 .................................................................... second allele of the VCP gene
Vg ................................................................................................... vitellogenin
WCP .............................................................................. wheat carboxypeptidase
YP1 .............................................................................. Drosophila yolk protein 1
YP2 .............................................................................. Drosophila yolk protein 2

xi

CHAPTER 1

INTRODUCTION

1 . Historical Perspective:

Mosquitoes are the most prominent insect pests of human populations
throughout the world. By acting as a biting nuisance and as vectors of human
disease, these animals take a great toll in both human health and financial
resources. In California, almost 55 million dollars per year are spent on
mosquito control and research for a region with a population of 22 million
people. An additional 45 million dollars are spent in Florida each year to combat
the problem. Worldwide, hundreds of millions of dollars have been spent in an
attempt to control mosquito populations to reduce malaria and other diseases
(Table 1) (McClelland, 1992).

Attempts to control the spread of mosquito-bome diseases have often
focused on control of insect populations. In the mid 1950's, a global malaria
eradication campaign was begun, largely funded and organized by the World
Health Organization. This campaign relied primarily on the use of DDT as an
insecticide and chloroquine to treat infected individuals. Initial results of the
campaign were encouraging; in India, the number cases of malaria fell from 75
million in 1953 to 50,000 in 1958. Similar results were obtained in other regions
of the world. However, as pesticide resistance grew in the mosquito population
and chloroquine resistance of the parasite increased, incidence of disease
rapidly rose to levels seen before the campaign began. Indeed, in 1977, 50
million cases of malaria were detected in India (McClelland, 1992).

In recent decades, much effort and emphasis has been placed on the
development of a vaccine against the malaria parasite. This search has been
largely unsuccessful thus far, with no substantially effective vaccine likely
available in the near future. Such a development would be highly beneficial to

people traveling into endemic areas or residents of cities in areas with high

Table 1: A sample of the costs of recent epidemics of mosquito-borne

diseases in several regions of the world. (From McClelland, 1992 ).

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malaria rates. However, in much of the malarious zones of the third world,
problems of poverty, shortages, communications, transportation and political
unrest make the feasibility of high levels of vaccination doubtful. Thus the
benefits of vaccination to a substantial portion of the at risk population may be
unrealistic (Anderson and May, 1991 ).

Numerous other illnesses are carried by mosquitoes in various parts of
the world. Mosquitoes from the genus Aedes carry the viruses responsible for
Dengue, yellow fever, and numerous varieties of encephalitis as well as the
parasite responsible for filariasis. Anopheles mosquitoes are most known as
malaria vectors, but also carry filariasis parasites. Control of major outbreaks
and epidemics of these diseases also depends on control of the vector
population. Because of the rapidly rising incidence of resistant populations of
mosquitoes to current pesticides and growing dislike of large scale application of
chemical insecticides, new and innovative control strategies are needed. The
development of such methods are dependent on basic research into the biology
of these insects. The rapidly developing field of molecular biology provides new

tools for use in this investigation.

2. Basic Physiology of Female Mosquito Reproduction:

In anautogenous (blood feeding) mosquitoes, egg production depends on
a blood meal. Over the lifetime of an individual female, multiple blood meals are
taken, one or more for each clutch of eggs. Pathogens are acquired by the
mosquito during initial blood meals and passed on to future hosts through
subsequent feeding. In this way, the propagation of disease causing organisms
and the spread of mosquito-bome diseases are both intimately linked to the
behavior and physiology of the female. For this reason, a great deal of research

has been conducted to build an understanding of how this process works in

6

hopes of developing ways to interrupt either the reproduction of the insect or the
propagation of the pathogen.

The cyclical nature of egg production in mosquitoes differs significantly
from that of Drosophila, in insect more familiar to geneticists. Where as in fruit
flies adult females produce eggs continuously, mosquitoes instead produce
large numbers of completely synchronous eggs at distinct time intervals. As a
result, the insect undergoes rather drastic, tightly controlled physiological
changes that facilitate large scale synthesis of the many components of the
eggs, followed by remodeling of the synthetic tissue for subsequent egg
producing cycles. This process, called vitellogenesis, is regulated by various
hormones that act specifically at different points in the cycle.

The major synthetic tissue during vitellogenesis is the fat body, the insect
analog to the vertebrate liver. Tissue of this type is found primarily in the
abdomen, arranged as sheets or lobes which are attached to the epidermis and
surround the midgut. Additional fat body cells are also found in the head and
thorax. The activity of this tissue during egg production can be divided into three
distinct stages: 1) a preparatory phase in which the female develops the
competence to produce eggs, 2) an intensely synthetic phase during which
large amounts of yolk protein molecules are produced and secreted into the
hemolymph for subsequent storage in the rapidly maturing oocytes and 3) a
termination phase where the biosynthetic organelles involved in yolk protein
production are broken down by Iysosomes (for review, see Raikhel, 1992).

The preparatory phase of development is absolutely necessary for
vitellogenesis to occur. This stage lasts for about three days beginning from the
time the adult ecloses from the pupa. During this time, the insect is unwilling to
take a blood meal and cannot produce eggs. Two major cellular events take

place in the fat body during this period: 1) an increase in ploidy (Dittman et al.,

7

1989), and 2) a proliferation of ribosomes (Raikhel and Lea, 1990). These
processes appear to be controlled by juvenile hormone Ill (JHIII)(Fig. 1). The
titer of this hormone rises over the first 2 days after eclosion, then slowly
declines over the next five days in females not given a blood meal (Shapiro et
al., 1986). The rate of RNA synthesis and the amount of ribosomal RNA in fat
body cells as well as synthesis of DNA directly follow the increase in JHlll. After
three days of preparatory development, the insect goes into a state of arrest.
During this time her reproductive cycle remains static, without major changes
until a blood meal is obtained.

With the acquisition of a blood meal, the synthetic phase of vitellogenesis
begins. During this time the fat body produces massive quantities of yolk
proteins which are secreted into the hemolymph and taken up by the developing
oocytes. This period lasts from the time of the blood meal until approximately 36
hrs later, when yolk protein synthesis and uptake have ceased (Koller et al.,
1989). During this time, the levels of JHIII fall to very low levels, while the titer of
the steroid hormone 20-hydroxyecdysone (20E)(Fig. 1) increases dramatically
(Shapiro et al., 1986; Hagedom, 1983). The amount of 20E peaks at 18-24 hrs
after a blood meal, then begins to fall rapidly in the later stages of the synthetic
phase. This rise and fall in 20E titer is correlated with the changes in the rate of
yolk protein synthesis, leading to the initial hypothesis that 20E controls
production of the yolk proteins, probably at the level of transcription (Fallon et
al., 1973). This topic has been the subject of a considerable amount of
research, and remains unclear.

Once the titer of 20E has fallen to pre-blood meal levels, the termination
phase of vitellogenesis begins. This stage lasts from approximately 30 hrs to 48
hrs post blood meal, after which the mosquito is ready to lay eggs and begin the

entire cycle anew. Termination involves two distinguishable steps: 1) turning off

Fig. 1: The structure of the two major hormones controlling mosquito

vitellogenesis. (Oberlander, 1985).

HO

HO

OH
OH

OH

 

 

20-hydroxyecdysone

Juvenile Hormone Ill

10

the expression of the yolk protein genes and 2) destruction of the biosynthetic
machinery (rough endoplasmic reticulum, Golgi complexes and secretory
vesicles) responsible for yolk protein secretion (Raikhel 1986a; Raikhel, 1986b).
This is accompanied by a large proliferation of Iysosomes within the cells. The
fat body cells are remodeled and readied to re-enter the preparatory phase to
initiate a new round of the reproductive cycle. This entire cycle is summarized in
Fig. 2.

3. Control of Yolk Protein Synthesis:

Perhaps the most dramatic aspect of the vitellogenic cycle is the transition
of the fat body from its state-of-arrest, to 36 hrs of extremely intense yolk protein
synthesis, then its subsequent return to a non-synthetic state. The exact nature
of how this tightly regulated process is controlled has been the subject of a great
deal of speculation and research. The initial hypothesis that the titer of 20E
directly controls the transcription of the yolk protein genes and, as a result, the
rate of yolk protein synthesis, was simple and intuitive. To test this hypothesis, a
number of in vivo and in vitro experiments were done. When fat bodies of
mature females were cultured in vitro, physiological doses of 20E were found to
stimulate yolk protein synthesis (Fuchs and Kang, 1981; Hagedom, 1985).
However, in vivo injections of 20E required large doses to generate similar levels
of yolk protein production (Fuchs and Kang, 1981; Lea, 1982). In addition, yolk
protein induction at both the protein and mRNA levels is detectable in females
only 1 hr after a blood meal, significantly before increases in 20E titers can be
measured (Raikhel and Lea, 1983; Racioppi et al., 1986). Thus, 20E is clearly a
significant factor in the initiation of vitellogenesis, however, exactly how this

hormone exerts control over its target genes remains unclear.

11

Fig. 2: Summary of events during the first cycle of egg maturation in the
anautogeneous mosquito, Aedes aegypti. (A) Hormonal Events. EDNH, egg
development neurosecretory hormone; JHlll, juvenile hormone lll; NHRF,
neurosecretory hormone-releasing factory; OEH, ecdysteroidogenic hormone;
OSH, oostatic hormone; 20E, 20-hydroxyecdysone. (B) Events in the fat body.
DNA, rate of DNA synthesis; VCP, rate of synthesis of vitellogenic
carboxypeptidase; Vg(s), rate vitellogenin synthesis. (C) Events in the ovarian
primary follicle. CV, number of coated vesicles per unit of the oocyte cortex; CH,
chorion deposition; FC, follicle cell; 00, oocyte yolk length; SPC, closing of
interfollular spaces; SPO, openning of interfollicular spaces; VE, vitelline

envelope formation; Vg(e), rate of V9 endocytosis. (From Raikhel, 1992).

12

 

 

 

 

 

 

 

 

 
   

 

 

 

 

 

 

 

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13

4. The Ashbumer Model of 20-hydroxyecdysone Control of Gene
Expression:

Ashbumer and his colleagues proposed a model to describe how 20E
regulates gene expression in Drosophila salivary glands (Ashbumer et al.,
1974). Working with polytene chromosomes that were exposed to 20E under a
variety of conditions, they observed puffing at the loci of genes specifically
regulated by this hormone. In this model, 20E enters the cell and is bound by its
receptor. The activated hormone/receptor complex then differentially regulates
two classes of genes. The first class, referred to as the early genes, is a small
set of loci encoding transcription factors that repress their own transcription and
induce transcription of the late genes. The late genes are a large class of loci
that represent the bottom of the hierarchy. The model provides for regulation at
the levels of the hormone, the receptor, the early genes and the late genes.
This control is exhibited in sex-, tissue- and stage-specific ways.

In recent years, a number of molecular studies have been published that
support this model of 20E action in Drosophila salivary glands. Three of the
early puffing loci have been cloned and analyzed. These include the E75 gene
(Segraves and Hogness, 1990), the E74 gene (Thummel et al., 1990) and a
member of the Broad-Complex (DiBello et al., 1991). As predicted, transcription
of each of these genes is induced by 20E in the initial response, then
subsequently repressed as the late genes are activated. These genes encode
DNA binding proteins that show significant homology to transcription factors. In
addition, E74 and E75 proteins were shown to bind to both the early and late
classes of ecdysone responsive puff sites on the polytene chromosomes. These
observations are consistent with the idea that these early gene products repress

their own expression while inducing late gene transcription.

14

In the mosquito, such detailed studies of the action of 20E have not been
done. During vitellogenesis, control of the yolk proteins by 20E could follow two
potential pathways. The hormone, bound by its receptor, may bind directly to
the control regions of these genes, inducing high levels of expression.
Alternatively, it may act through a hormone response cascade, with the yolk
protein genes at the bottom of the hierarchy. In this way, the yolk protein genes
would represent "late" loci in the Ashbumer model. Either way, the control of
expression of these genes is likely to be quite complex, with numerous factors

determining the sex-, stage-, and tissue-specific control exhibited.

5. Yolk Protein Expression in Drosophila:

Considerable research has been done on the regulation of the yolk
protein genes in Drosophila. Experiments using hormone injections and organ
culture have shown that yolk protein synthesis occurs in response to either 20E
or JHlll (Jowett and Postlethwait, 1980; Bownes, 1982; Bownes et al., 1983). In
fruit flies, however, yolk proteins are produced by both the fat body and the
follicle cells of the ovary. In addition, males can be induced to produce yolk
proteins by injections of 20E (Kozma and Bownes, 1986). The sexual
differentiation pathway has also been implicated in the control of yolk protein
synthesis. Genetic analysis using mutants of the sex determination regulatory
gene transformer-2 have shown that proper expression of this gene is required
for expression of the yolk protein genes (Belote et al., 1985). The nutritional
status of the female also influences yolk protein production (Bownes and Blair,
1986).

Three yolk proteins have been found in Drosophila, named YP1 (46 kDa),
YP2 (45 kDa) and YP3 (44 kDa) (Warren and Mahowald, 1979). The proteins

are encoded by different, single copy genes, all located on the X-chromosome

15

(Barnett et al., 1980; Barnett and Wensink, 1981 ). YP1 and YP2 are divergently
transcribed, sharing a 1227 bp upstream regulatory region, while YP3 is located
at least 106 bp away. All three genes have been cloned and sequenced, and
considerable research has been done on their regulatory regions. Using P-
element mediated germline transformation, deletion analysis and site-directed
mutagenesis, several specific regulatory elements have been defined. These
include a 127 bp sequence that induces fat body specific expression of a
reporter gene (Shepherd et al., 1985; Garabedien et al., 1986), a 301 bp region
directing ovarian specific expression (Logan et al., 1989) and several specific
binding sites for a member of the sex determination pathway, doublesex
(Coshigano and Wensink, 1993). In addition, binding sites for two tissue-
specific transcription factors, BBF-2 (a member of the CREB/ATF family of
transcriptional regulatory proteins) and AEF-1 (adult enhancer factor 1) have
been identified between YP1 and YP2 (Falb and Maniatis, 1992; Abel et al.,
1992) This research provides a model for comparison when similar regulatory

regions in mosquito become available.

Yolk Protein Expression in Aedes aegyptl:

The study of vitellogenesis in mosquitoes is most advanced in the species
Aedes aegypti. This species is easily reared in a laboratory setting and provides
the best model for studying the physiology and molecular biology of mosquitoes
in general. Most research has centered on synthesis and secretion of the most
abundant yolk protein, termed vitellogenin (Vg). This is a 380 kDa protein
produced exclusively in the fat body and selectively stored in the developing
oocytes. The protein consists of 2 subunits, one of 200 kDa and the second of
66 kDa (Raikhel and Bose, 1988). Synthesis, processing and secretion of this

protein has been extensively studied. Recently, a second, much smaller yolk

16

protein was observed that was produced in a manner identical to V9 (Hays and
Raikhel, 1990). Unlike Vg, this protein is not phosphorylated or sulfated, and
shows small levels of glycosylation. Because of its smaller size and simpler
post-translational modifications, this 53 kDa protein has been considered a more
convenient marker for molecular studies of protein processing in the fat body
and internalization by the oocyte.

Research into yolk protein production at the gene level has been limited.
A multigene family consisting of 5 genes encoding Vg has been identified
(Hamblin et al., 1987). Four of these are thought to be actively transcribed and
all hybridize to a 6.5 kb mRNA present only in vitellogenic fat bodies.
Preliminary sequence data from the regulatory region of one of these gene has
identified a number of elements resembling a TATA box, a putative capsite and
sequence motifs similar to hormone response elements (Hagedom,
unpublished). Complete analysis of the structure of these genes is somewhat
cumbersome due to the large size of the coding region and the number of copies
in the genome. This combined with the lack of an expression system for
vitellogenic genes have slowed research in this area. As a result, nothing has
been published regarding the genes for A. aegypti Vg since their initial
identification. Analysis of the locus for the recently identified 53 kDa yolk protein
may represent an alternative research pathway, providing a smaller, perhaps
single copy gene for analysis.

Beyond the basic research interests for investigating the regulatory
regions responsible for yolk protein expression, there is also a desire to obtain
such DNA sequences for use in possible genetic engineering of mosquitoes
against disease causing parasites (Aldhous, 1993). Numerous laboratories are
currently attempting to develop methods of germline transformation of disease-

vector species, as well as finding ways to move the genes of interest throughout

17
a natural population (Kidwell and Ribeiro, 1992; Ribeiro and Kidwell, 1994).

Candidate parasite-inhibiting genes for use in such a strategy are also being
developed (Huber et al., 1993). When potential anti-parasite genes have been
found, powerful promoters and regulatory DNA sequences will be needed to
express these genes in such a way as to make them effective against the
disease causing organism. Elements that control yolk protein gene transcription
may provide the regulatory sequences desired. Yolk protein genes are
expressed at extremely high levels in the major synthetic organ of the adult, at
the specific time period when the insect is likely to come into contact with the
parasite. Thus, the regulatory region of the gene for V9 or the 53 kDa protein
could be of great value to the development of a biological control strategy

involving mosquito genetic engineering.

18
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Aldhous, P., 1993, Malaria: focus on mosquito genes, Science, 261: 546-548.

Anderson, RM. and May, RM, 1991, Infectious Diseases of Humans, Oxford
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Ashbumer, M., Chihara, C., Meltzer, P. and Richards, 6., 1974, Temporal
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Barnett, T., Pachl C., Gergen, JP. and Wensink, PC, 1980, The isolation and
characterization of Drosophila yolk protein genes, Cell 21: 729-738.

Barnett, T. and Wensink, PC, 1981, Trancription and translation of yolk protein
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Belote, J.M., Handler, A.M., Wolfner, M.F., Livak, K.J. and Baker, 88., 1985,
Sex-specific regulation of yolk protein gene expression in DroSOphila. Cell
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Bownes, M., 1982, The role of 20-hydroxyecdysone in yolk-polypeptide
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Bownes, M. and Blair, M., 1986, The effects of a sugar diet and hormones on the
expression of the Drosophila yolk-protein genes, J. Insect Physiol. 32:
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Bownes, M., Blair, M., Kozma, R. and Dempster, M., 1983, J. Embryol. Exp.
Morph. 78: 249-268.

Coschigano, KT. and Wensink, PC, 1993, Sex-specific transcriptional
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DiBello, P.R., Withers, D.A., Bayer, C.A., Fistrom, J.W. and Guild, GM, 1990,
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Dittman, F., Kogan, PH, and Hagedom, H.H., 1989, Ploidy levels and DNA
synthesis in the fat body cells of the adult mosquito, Aedes aegypti: the
role of juvenile hormone, Arch. Insect Biochem. Physiol. 12: 133-143.

Falb, D. and Maniatis, T., 1992, A conserved regulatory unit implicated in tissue-
specific gene expression in Drosophila and man, Genes Develop. 6: 454-
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Fallon, AM., Hagedom, H.H., Wyatt, GR, and Laufer, H., 1974, Activation of
vitellogenin synthesis in the mosquito Aedes aegypti by ecdysone, J.
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Flanagan, TR, and Hagedom, H.H., 1977, Vitellogenin synthesis in the
mosquito: the role of juvenile hormone in the development of
responsiveness to ecdysone, physiol. Entomol. 2: 173-178.

Fuchs, MS, and Kang, S-H., 1981, Ecdysone and mosquito vitellogenesis: a
critical appraisal, Insect Biochem. 11: 627-633.

Garabedien, M.J., Shepherd, B.M. and Wensink, PC, 1986, A tissue-specific
transcription enhancer from the Drosophila yolk protein 1 gene, Cell 45:
859-967.

Hagedom, H.H., 1983, The role of ecdysteroids in the adult insect, in Downer,
R.G.H., and Laufer, H., (eds): Endocrinology of Insects, Alan R. Liss, Liss,
Inc., New York, pp. 271-304.

Hagedom, H.H., 1985, The role of ecdysteroids in reproduction, in Kerkut, GA.
and Gilbert, L.I., (eds): Comprehensive Insect Physiology, Biochemistry,
and Pharmacology, vol. 8, Pergamon Press, Oxford, pp.205-261.

Hagedom, H.H., Fallon, AM, and Laufer, H., 1973, Vitellogenin synthesis by the
fat body of Aedes aegypti, Develop. Biol. 31: 285-294.

Hamblin, M.T., Marx, J.L., Wolfner, ME, and Hagedom, H.H., 1987, The
vitellogenin gene family of Aedes aegypti, Mem. Inst. Oswaldo Cruz 82:
109-1 14.

Hays, AR, and Raikhel, AS, 1990, A novel protein produced by the
vitellogenic fat body and accumulated in mosquito oocytes, Roux's Arch.
Develop. Biol. 199: 114-121.

20

Huber, M., Cabib, E., and Miller, L.H., 1991, Malaria parasite chitinase and
penetration of the mosquito peritrophic membrane, Proc. Natl. Acad. Sci.
USA 88: 2807.

Kidwell, MG. and Ribeiro, J.M.C., 1992, Can transpcsable elements be used to
drive disease refractoriness into vector populations?, Parasitol. Today 8:
325.

Kozma, R. and Bownes, M., 1986, Yolk protein induction in males of several
Drosophila species, Insect Biochem. 16: 263-271.

Lea, AD, 1982, Artifactual stimulation of vitellogenesis in Aedes aegypti by 20-
hydroxyecdysone, J. Insect Physiol. 28: 173-176.

Logan, SK, Garabedien, M.J. and Wensink, PC, 1989, DNA regions that
regulate the ovarian transcriptional specificity of Drosophila yolk protein
genes, Genes Develop. 3: 1453-1461.

Ma, M., Zhang, J-Z., Gong, H., and Gwarz, R., 1988, Permissive action of
juvenile hormone on vitellogenin production by the mosquito, Aedes
aegypti, J. Insect Physiol. 34: 593-596.

McClelland, G.A.H., 1992, Medical Entomology: an Ecological Perspective,
University of California Press, Davis, California, USA

Oberlander, H., 1985, Hormonal action during insect development, In: Blum M.
(ed), Fundamentals of Insect Physiology, John Wiley and Sons, inc., New
York, 51 3-514.

Postlethwait, J.H. and Jowett, T., 1980, Genetic analysis of the horomonally
regulated yolk polypeptide genes in D. Melanogaster, Cell 20: 671 -678.

Racioppi, J.V., Gemmill, R.M., Kogan, P.H., Calvo, J.M., and Hagedom, H.H.,
1986, Expression and regulation of vitellogenin messenger RNA in the
mosquito Aedes aegypti, Insect Biochem. 16: 255-262.

Raikhel, A.S., 1986a, Role of Iysosomes in regulating of vitellogenin secretion in
the mosquito fat body, J. Insect Physiol. 32: 597-604.

Raikhel, A.S., 1986b, Lysosomes in the cessation of vitellogenin secretion by the
mosquito fat body: selective degradation of Golgi complexes and
secretory granules, Tissue Cell 18: 125-142.

21

Raikhel, AS, 1992, Vitellogenesis in mosquitoes, in: Advances in Disease
Vector Research, Springer-Verlag, New York, Inc., vol. 9, pp. 1 -39.

Raikhel, AS, and Bose, SC, 1988, Properties of mosquito yolk protein: a study
using monoclonal antibodies, Insect Biochem., 18: 565-575.

Raikhel, AS, and Lea, A0, 1983, Previtellogenic development and vitellogenin
synthesis in the fat body of a mosquito: an ultrastructural and
immunocytochemical study, Tissue Cell 15: 281 -300.

Raikhel, AS, and Lea, A0, 1990, Juvenile hormone controls previtellogenic
proliferation of ribosomal RNA in the mosquito fat body, Gen. Comp.
Endocrinol. 77:423-434.

Ribeiro, J.M.C. and Kidwell, MG, 1994, Transposable elements as population
drive mechanisms: specification of critical parameter values, J. Med. Ent.
31: 10.

Seagraves, WA, and Hogness, D., 1990, the E75 ecdysone-inducible gene
responsible for the E75B early puff in Drosophila encodes two new
members of the steroid receptor superfamily, Genes Develop. 4: 204-219.

Shapiro, A.B., Wheelock, G.D., Hagedom, H.H., Baker, F.C., Tsai, L.W., and
Schooley, DA, 1986, Juvenile hormone and juvenile hormone esterase
in adult females of the mosquito Aedes aegypti, J. Insect Physiol. 32: 867-
877.

Shepherd, 8, Garabedien, M.J., Hung, M.-C. and Wensink, PE, 1985,
Developmental control of Drosophila yolk protein 1 gene by cis-acting
DNA elements, Cold Spring Harbor Symp. Quant. Biol. 50: 521 -526.

Thummel, C.S., Burtis, KC, and Hogness, OS, 1990, Spatial and temporal
patterns of E74 transcription during Drosophila development, Cell 61:
101-1 1 1.

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characterization of the three major yolk polypeptides from Drosophila
melangaster, Proc. Natl. Acad. Sci. USA 76: 2848-2852.

22
CHAPTER 2‘

AN EXTRAOVARIAN PROTEIN ACCUMULATED IN MOSQUITO
OOCYTES IS A CARBOXYPEPTIDASE ACTIVATED
IN EMBRYOS

*Originally published in Proceedings of the National Academy of Sciences
Reference: Cho, W.-L., Deitsch, KW., and Raikhel, AS. (1991) Proc. Natl.
Acad. Sci, 88, 10821-10824.

23
ABSTRACT

We report a phenomenon previously unknown for oviparous animals; in
Aedes aegypti mosquitoes a serine carboxypeptidase is synthesized
extraovarially and then internalized by oocytes. The cDNA encoding mosquito
vitellogenic carboxypeptidase (VCP) was cloned and sequenced. The VCP
cDNA hybridizes to a 1.5 kilobase mRNA present only in the fat body of
vitellogenic females. The deduced amino acid sequence of VCP shares
significant homology with members of the serine carboxypeptidase family.
Binding assays using a serine protease inhibitor, [3deiisopropyl
fluorophosphate, showed that VCP is activated in eggs at the onset of embryonic
development. Activation of VCP is associated with the reduction in its size from
53 kDa (inactive proenzyme) to 48 kDa (active enzyme). The active, 48 kDa,
form of VCP is maximally present at the middle of embryonic development and

disappears by the end.

24
INTRODUCTION

In oviparious animals, development of an embryo depends upon
utilization of yolk proteins accumulated by oocytes during vitellogenesis. In most
oviparous animals, e.g., nematodes, arthropods, and vertebrates, the task of
producing massive amounts of yolk protein precursors is performed by
extraovarian tissues (1-5). The developing oocytes of these animals are highly
specialized for the specific accumulation of extraovarian yolk protein precursors.
The process of receptor-mediated endocytosis by which yolk protein precursors
are accumulated in oocyte yolk bodies is well characterized for insects and
vertebrates (6,7). In contrast, the mechanisms of degradation of yolk proteins
have received considerably less attention. Several enzymes that participate in
hydrolysis of yolk proteins in insect embryos have been recently described (8-
11). However, the origin of these enzymes and the time of their delivery into
yolk bodies are unknown.

In this paper, we report a biological phenomenon previously unknown for
insects or other oviparous animals. In vitellogenic female mosquitoes, an
extraovarian tissue, the fat body, synthesizes not only the yolk protein precursor
vitellogenin but also a proenzyme of a serine carbosypeptidase. We have
named it vitellogenic carboxypeptidase (VCP). This enzyme is internalized by
developing oocytes, accumlated in yolk bodies, and activated during embryonic

development.

25
MATERIALS AND METHODS

Animals:
Aedes aegypti mosquitoes were reared as described by Hays and Raikhel
(12). The embryonic stages of A. aegypti were determined according to

Raminani and Cupp (13).

Cloning and Sequencing of cDNA:

A cDNA clone encoding VCP was isolated by immunoscreening of a i.
gt11 library generated from fat bodies of vitellogenic female mosquitoes. This
cDNA clone, pVCP-1, was used to isolate another cDNA clone, pVCP-2,
encoding the 5'-end 1020 base pairs (bp) of the VCP cDNA from the same
library. The pVCP-2 cDNA clone was sequenced in both direction with the
dideoxy chain termination method (ref. 14, pp. 13.65-13.69).

The missing part of the 3'-end sequence was obtained by the polymerase
chain reaction, using 20 nucleotides at the 3'end of pVCP-1 and (dT)17 as
primers and including the Xba I site at the 5' ends of both primers (15).
Amplification was achieved by using cDNA reverse transcribed from 20 pg of
total RNA prepared from whole bodies of mosquitoes 24 hrs after a blood meal
as templates. The specific amplified band, identified by Northern blot
hybridization to 1.5 kilobase (kb) mRNA, was subcloned in pUC119 for
sequencing. Like the pVCP-2 clone, the 3'-end VCP clone was sequenced in
both directions.

Protein Purification for Partial Sequencing:
Mosquito ovaries, 48 hr after a blood meal, were homogenized. in 20mM
Tris-HCI, pH 7.5/150 mM NaCl/0.02 % sodium, azide. VCP was isolated from

26

the protein extract by anion-exchange, affinity, and hydrophobic
chromatographic steps using DEAE-Sepharose CL-4B, Concanavalin A, and
phenyl-Sepharose columns, respectively. A fraction with nearly pure protein was
resolved by SDSIPAGE and subjected to peptide sequencing using the method
and facilities described by Cho et al. (16).

Purification and Analysis of RNA:

Isolation of total and polyadenylated RNA [poly(A)"' RNA] from mosquito
tissues was performed as previously described (17).

In vitro translation of mosquito poly(A)+ RNA was performed as described
by Bose and Raikhel (17), using rabbit reticulocyte lysate (Promega). The VCP
precursor was identified in in vitro translation products of poly(A)+ RNA by
immunoprecipitation with anti-VCP polyclonal antibodies (12).

The 840-bp insert from pVCP-1, a putative VCP cDNA clone, was used
for hybrid selection of VCP mRNA from fat body poly(A)+ RNA according to
Sambrook et al. (ref. 14, pp. 8.50-8.51). The hybrid-selected mRNA was
translated in vitro in rabbit reticulocyte lysate and the protein product was
resolved by SDSIPAGE. The pUC119 plasmid without an insert was used for
hybrid selection of mRNA as control.

For Northern analysis, total RNA was separated by 1.2 %
forrnaldehydelagarose gel electrophoresis, transferred to nitrocellulose paper,
and hybridized first to a 32P-Iabeled 840-bp VCP cDNA insert from pVCP-1
under high-stringency conditions. RNA hybridized to the VCP cDNA probe was
visualized by autoradiography of the blot. The probe was then stripped from the
blot (ref. 14, p.958) and the blot was rehybridized with a 32P-Iabeled 1.8-kb
fragment of vitellogenin gene A1 (gift of H.H. Hagedom, University of Arizona)

under high stringency conditions.

27

Immunoblot Analysis:
Preparation of proteins, SDSIPAGE, and immunoblotting were performed

as described by Hays and Raikhel (12).

Binding to Protein Inhibitor:

Protein extracts, prepared as for immunoblotting but without any protease
Inhibitors, were incubated with [3H]diisopropyl fluorophosphate ([3H]DFP;
DuPont) at 0.1 uCi/ul (1 uCi = 1 kBq) for 1 hr at room temperature. These
mixtures were then separated by SDSIPAGE under reducing conditions and
processed for fluorography.

Analysis of Deduced Amino Acid Sequence:

Analysis of the deduced amino acid sequence was performed by using
the FASTA program (University of Wisconsin Genetics Computer Group
software) according to the algorithm of Lipman and Pearson (18). The deduced
amino acid sequence of VCP was compared with amino acid sequences from the
National Biomedical Research Foundation Protein Data Base (release 21.0) and
with deduced amino acid sequences translated from GenBank (release 66.0)
and European Molecular Biology Laboratory (release 19.0) data bases. The
initial score was calculated from the best subsequence alignment of two
sequences. The optimized score, calculated by considering insertions or
deletions, gives a better indication about functional relation between proteins.
The statistical significance of the scores was evaluated by 2 value [(similarity
score- mean of random scores); 2 > 10 indicates statistical significance]. The
similarities between VCP and other sequences were calculated as the
percentage of amino acid number having identical residues or functional

substitutions relative to the total number of amino acids in VCP (441 ).

28

RESULTS AND DISCUSSION

Recently, a female-specific protein (M = 53,000), initially called 53KP,
has been found in A. aegypti. Like vitellogenin, this protein is synthesized by the
fat body of vitellogenic females under the control of 20-hydroxyecdysone. The
kinetics of 53KP production by the vitellogenic fat body is also similar to that of
vitellogenin: it is produced as early as 4 hr and reaches its peak near 24 hr after
the initiation of vitellogenesis. Synthesis then drops to low levels by 36 hr and
declines to background levels by 48 hr. This protein is secreted to the
hemolymph and is selectively accumulated in yolk bodies of developing oocytes
(12). Data presented here show that this protein (VCP) is a serine
carboxypeptidase.

The cDNA encoding VCP was cloned by a combination of
immunoscreening of a kgt11 cDNA library and the polymerase chain reaction.
The identity of the cDNA was confirmed by direct sequencing of the N terminus
of the purified VCP and by in vitro translation of hybrid selected mRNA (data not
shown).

Northern blot analysis demonstrated that transcription of the VCP mRNA
is limited to female fat bodies and is intiated after a blood meal. Similar to
vitellogenin mRNA, the VCP mRNA in the fat body is maximal at the peak of the
protein production, 24 hr after a blood meal (figs. 1 and 2).

29

Fig. 1: Northern analysis of sex- and stage-specific expression of the VCP
mRNA transcript. Total RNA was extracted from whole bodies of male
mosquitoes (lane 1) and fat bodies of female mosquitoes before a blood meal
(lane 2) or 24 and 48 hr after a blood meal (lanes 3 and 4 respectively). An RNA
ladder (BRL) was resolved in lane M. (A) Agarose gel stained with ethidium
bromide. (B) Hybridization with 0.84-kb fragment of VCP cDNA. (C)
Hybridization with 1.8 kb fragment of mosquito vitellogenin A1 gene.

30

M123412341234

 

31

Fig. 2: Northern blot analysis of tissue-specific expression of the VCP
mRNA transcript. Total RNA was extracted from ovaries (lane 1), fat bodies
(lane 2), and midgut (lane 3) of female mosquitoes 24 hr after a blood meal. An
RNA ladder (BRL) was resolved in lane M. (A) Agarose gel stained with
ethidium bromide. (B) Hybridization with 0.84-kb fragment of VCP cDNA.

32

An 1 2 3 4 1 2 .3 4

(kb)

1l5\g
15/”
4.4 —

ZA——
L4-—

.24-—

 

33

The sequence of the full length cDNA encoding VCP, confirmed by
sequencing of cDNA clones in both directions, is presented in Fig. 3. The size
of the VCP mRNA of 1.5 kb, estimated by Northern blot analysis, is in agreement
with the 1511-bp mRNA estimated from the cDNA sequence. The VCP cDNA
has a single open reading frame that encodes a protein of 441 amino acids with
a deduced molecular mass of 50,153 Da. Cell-free translation of VCP mRNA
revealed that the VCP precursor is a 50-kDa polypeptide (data not shown).

Hydropathy analysis (19,20) of the deduced amino acid sequence has
shown that VCP exhibits properties typical of a secretory protein. The amino
acid sequence of VCP has only one potential glycosylation site, at position Asn-
135. Glycosylation of VCP by mannose polysaccharide, which accounts for
about 2 kDa of the molecular mass, was demonstrated previously (12).

In other insects, small extraovarian proteins are also involved in formation
of yolk protein reserves. Microvitellogenin (31 kDa) of Manduca sexta and
Hyalophora cecropia and the 30-kDa protein of Bombyx man are produced by
the fat body and deposited in oocytes (21-23). The nucleotide sequence of the
Manduca microvitellogenin cDNA has 70% similarity to the cDNA sequence
coding for the Bombyx 30-kDa protein, indicating a close evolutionary
relationship between these proteins (24-26).

Comparison of amino acid sequences has not revealed any similarity
between mosquito VCP and either Manduca microvitellogenin or Bombyx 30-
kDa protein. Unexpectedly, the VCP sequence exhibited significant similarity
with members of a family of serine carboxypeptidases (Table 1). The similarity
between the amino acid sequences of mosquito VCP and these
carboxypeptidases is highest at the N-terminal portion, which includes two
conserved domains (27-32). The highest identity (28.1%) was found in a stretch
of 392 amino acids of VCP and wheat carboxypeptidase Y homolog (WCP)

Fig. 3: Nucleotide and deduced amino acid sequences of mosquito VCP.
The amino acid sequence matching the N terminus determined from purified
VCP is underlined by a solid line. The signal peptide is boxed. The circled
amino acid (N) is a potential glycosylation site. A putative polyadenylation

signal, AATAAA, is underlined by a broken line.

H

M H H

OJ

up mm ON H0 00 up mm wN H0 00 db mm OM Hm
OH OH OH OH OH OH OH OH OH OH OH OH OH OH

\0 NCO le in Mm H01 I—‘U'l I—‘b l—‘b I-JLJ

H

p

H H H
cu AN UN wH uo mo mm M

H
UN I-‘U‘ \DO NI-D U‘m UN I-‘O‘ \00

OH OH OH OH OH OH OH OH

H
L.)
m
H

1441
1501

35

 

TTCCCACTCGTTCTAAAGTCTAATCGAACAACC TQGTGAAAT‘I‘CCATTTACTACTCCTG
V K P H I L V I;

ATTGCGTTCACCTCCTATACA'I‘GCAGCGACGCAACCTTATGGAATCCGTACAACAAGCTA
I A F' 'l' C Y 'I' C S D A T L W N P Y K K L

ATGCGAGGATCCGCGTCTCCTCGTCGTCCACGTGAAAGTGGTGAACCTTTGTTCCTGACT
H R G S A S P R R P G E S G E P L F L T

CCACTGTTGCAGGATGGCAAAATTCAAGAGGCTCGCAACAAACCCCCCCTCAACCATCCC
P L L Q D G K I E E A R N K A R V N H P

ATCTTCAG CTCACTCGAAAGCTACTCCGCTTTTATCACCCTTG ATCCCAAG CACAACTCC
M L S S V E S Y S C I" H T V D A K H N S

AATTTGTTCTTCTGGTATGTTCCACCGAAGAACAACCGCGAACAAGCGCCCATTCTTCTT
NLFFWYVPAKNNREQAPILV

TGGCTGCAAGGAGGTCCAGGTGCGTCATCCCTGTTTGGAATGTTCGAAGAGAATGGACCG
W L Q G G P G A S S L F G H P E E N G P

TTCCATATTCACAGG CAACTCAGTGAAGCAACGTGAATATTCCTGCCATCACAACCAT
F H I H R N S V K Q R E Y S W H Q N

CACATGATCTACATCGATAATCCAGTTGCAACGGGATTCAGTTTCACCGATAGCCATGAA
H H I Y I D N P V G T G F S F T D S D E

 

GGTTACTCCACCAACGAAGAACACGTGGGTGAAAATCTGATGAAATTCATTCAACAATTC
GYSTNEEHVGENLHKPIQQF

TTCGTGCTGTTCCCCAATCTGTTGAAGCATCCATTCTACATCTCCGGTGAATCTTATGGT
F V L F P N L L K, H P F Y I S G E S Y C

GGAAAGTTTGTTCCTGCCTTCGGTTATGCCATCCACAACTCCCAAAGCCAACCTAAAATC
G K F V P A F G Y A I H N S Q S Q P K I

AATCTGCAAGGATTGGCCATTGGTCATGGCTACACTGATCCCCTGAACCAACTTAACTAC
NLQGLAIGDGYTDPLNQLNY

GGAGAATACCTGTATGAGCTGGGCCTGATCGATTTGAACGGAAGAAAGAAGTTCGACGAG
GEYLYELCLIDLNGRKKFDE

GATACGGCTGCTGCCATCGCCTGTGCCGAACGTAAGGACATGAAGTGCGCCAACCGCCTT
DTAAAIACAERKDHKCANRL

ATCCAAGGTCTGTTCGATGGACTCGATGGACAGGAATCGTACTTCAAGAAGCTCACCGGA
IQGLFDGLDGQESYFKKVTG

TTCTCGTCCTACTACAACTTCATCAAGGGAGACGAGGAGAGCAAACAGGATAGTGTCCTG
F S S Y Y N F I K G D E E S K Q D S V L

ATGGAGTTCCTCAGCAACCCGGAGGTACGTAAGGGCATCCACGTTGGTGAACTGCCGTTC
H E F L S N P E V R K G I H V G E L P F

CACGACTCTGACGGTCACAACAAGGTCGCGGAAATGCTGTCCGAAGACACTCTGGACACC
HDSDGHNKVAE TLDT'

HLSED

GTGGCTCCATGGGTCAGCAAGCTGCTCTCGCACTACCGCGTGCTGTTCTACAACGGTCAG
VAPHVSKL'LSHYRVLFYNGQ

TTGGACATCATCTGCGCCTACCCGATGACGGTCGACTTCCTGATGAAGATGCCTTTCGAT
LDIICAYPHTVDFLHKHPFD

GGCGATAGCGAGTACAAGCGGGCCAATCGTGAGATCTACCGCGTGGATCGGAAATCGCCG
G D S E Y K R A N R E I Y R V D R K S P

GGTACAAGAAGCGGGCTGCTCGTCTGCAAGAGGTCCTGATCAGAAACCCCGGACACATGG
G T R S G L V V C K R C

TTCCGCGGGACCAGCCGAAATGGCCTTTCGACATCATCACCTCCTTCACTCACAAAAACT

ACTTGTCAGGGAAATGGTAATGATTTGGATGAAIAAAGCTTTAAGCTGTAATTAAAAAAA
AAAAAAAAAAA

M H H
on

L1

PJN 001 (DC (DA DOD NM 00‘ 030 OMB- bCD NM

Nd Mm Hm Hm HA Ho Hu
mo 00 00 mo 00 no 00 mo 00 mo 00 oo 00 mo 00

O ULD N10 moo his)
(DO 01:) AC)

H H H H H
bu AN um wH wo u

H
\00 \OO \00 \00 £00 \00 \00

NM 00" m0 0113 Am NM 00)

p...
L.)
(I)
O

1440
1500

Table 1. Homology of mosquito VCP to several serine carboxypeptidases.

37

TABLE 1. Homology of mosquito VCP to serine carboxypeptidases.

 

Score Z-value
I O I 0
Wheat: Carboxypeptidase Y homolog 292 523 45.4 81.1
Yeast: Carboxypeptidase Y 209 387 30.4 43.2
Human: Protective protein 194 422 38.4 59.2
Mouse: Protective protein 190 434 25.9 54.8
Yeast: KEXl carboxypeptidase 182 339 37.3 40.0
Barley: Serine. carboxypeptidase I I 139 394 18.5 42.9
Barley: Serine carboxypeptidase II 116 380 16.5 49.1

 

I -.initial score, 0 - optimized score, Z-value - statistical significance.

38

(Fig. 4). However, considering both identical and conservative replacements,
similarity between VCP and WCP is 62.3%. In mosquito VCP the conserved
domains are at positions Trp-110 to Ser—119 and Gly-205 to Gly-210 (Fig. 4).
The Ser-207 of the VCP second domain corresponds to Ser-257 of yeast
carboxypeptidase Y, in a sequence that was shown to be the catalytic center of
serine carboxypeptidases (33). In contrast to mosquito VCP and WCP (Fig. 4),
all other members of the serine carboxypeptidase family have three conserved
domains (27-32). Functional implications of these differences are not clear.
Unlike the situation for serine endopeptidases, the importance of three domains
for carboxypeptidase activity has not been established (34). Similar to all other
serine carboxypeptidases in the family (27-32), mosquito VCP does not have
any significant homology with any known sequences of trypsin-Iike serine
endopeptidases or esterases.

lntemalization of VCP in mosquito oocytes occurs without any changes in
its size (12). The molecular mass of VCP, however, decreases by 0.5-1.0 kDa at
the onset of embryonic development (Fig. 5). As embryogenesis progresses,
VCP undergoes further reduction in its size and another, immunologically related
48 kDa band appears (Fig. 5). By the end of embryonic development, VCP
degrades into smaller peptides, which disappear once the first-instar larva
hatches (Fig. 5).

Radiolabeled serine protease inhibitor, [3H]DFP, which binds to the active
center of serine proteases (35), binds weakly to VCP in oocytes (Fig. 5, lanes 1).
The intensity of binding to VCP, however, increases at the onset and reaches
maximum in the middle of embryogenesis, when the inhibitor binds to a VCP
band of 48 kDa (Fig. 5). The binding of DFP to VCP can be inhibited by prior
treatment of protein extracts with other serine protease inhibitors,

phenylomethylsulfonyl fluoride or leupeptin (data not shown). On the basis of

39

Fig. 4: Alignment of amino acid sequences of mosquito VCP and WCP.
Amino acid sequences deduced from corresponding cDNAs were aligned by
FASTA computer program. Vertical lines indicate identical residues and oolons
denote functional substitutions. Only portions of both amino acid sequences,
flanking the overlapping regions, are presented. Two conserved domains found
in sequences of serine carboxypeptidases are boxed. The serine protease

catalytic center is marked by an asterisk.

VCP

WCP

VCP

WCP

VCP

WCP

VCP

WCP

VCP

WCP

VCP

WCP

VCP

WCP

VCP

WCP

40

LWNPYKKLMRGSASPRRPGESGEPLFLTPLLQDGKIEEARNKARVNHPMLSSVESYSGFM

PGASSLFGMFEENGPFHIHRNNSVKQR

xiir.ii ...l lllll l .
til r "°l lllll l -i i- .

O O O . ‘ It:
0 O O O 2"?“
RLPNTHDARMFYFFFESRGKKED-PVVIWLTGGPGCSSELAVFYENGPFTIANNMSLVWN

....................................................

TVDAKHNSNLFFWYVPAKNNREQAPIL

 

  

EYSWHQNHHMIYIDNPVGTGFSFTDSDEGYSTNEEHVGENLMKFIQQFFVLFPNLLKHPF

ooo'oo oo'oo' 00"'I|oooo' o o 0'. '00.. 0'0' ': 'ooo'o'
ooc'oo ooloo' oo'|"‘oooo| o o o'o '00.. 0'0. 'OOOIO'

KFGWDKISNIIFVDPATGTGFSYSSDDRDTRHDEAGVSNDLYDFLQVFFKKHPEFVKNDF

..........................

 

J

LI---DLNGRKKFDEDTAAAIA-CAERKDMKC--ANRLIQGLFDGLDGQESYFKKVTGFS
" '0 ::' o :: '00 o :' .00 000...: 0'... 0'
" '0 o ‘0. o o. '0. 000'... 0'... o
LIQKADYDRINKFIPPCEFAIKLCGTDGKASCMAAYMVCNSIFNSI ------- MKLVGTK

SYYNFIKGDEE--SKQDSVLMEFLSNPEVRKGIHVGELPFHDSDGHNKVAEMLSEDTLDT
.l . . .l

NYYDVRKECEGKLCYDFSNLEKFFGDKAVRQAIGVGDIEFVSCS--TSVYQAMLTDWMRN

VAPWVSKLLSH-YRVLFYNGQLDIICAYPMTVDFLMKMPFDGDSEYKRANREIYRVDRKS
O. "O. 0".I... 'O".. O O. "
ll

l l
:: oo .‘oo o'|o|o'o 'o||oo o es :| 33.33:: ::: : 3

LEVGIPALLEDGINVLIYAGEYDLICNWLGNSRWVHSMEWSGQKDFAKTAESSFLVDDAQ

PGTRSGLVVCKRC

AGVLKSHGALSFLKVHNAGHHVPMDQPKAALEMLRRFTQGKLKESVPEEEPATTFYAA

81

141

150

201

210

257

270

311

323

369

381

428

441

441

499

41

Fig. 5: Immunoblot (A) and [3H]DFP binding (B) analyses of VCP during
mosquito embryonic development. Both panels contain protein extracts from
the following: lane 1, mosquito ovaries removed 24 hrs after a blood meal during
peak yolk accumulation; lane 2, 0- to 3-hr postoviposition eggs at the onset of
embryogenesis; lane 3, 44- to 47-hr postoviposition eggs during
midembryogenesis; lane 4, 94- to 97-hr postoviposition eggs at the end of
embryogenesis; lane 5, first-instar larva. The mosquito VCP bands are shown
by arrows. The high molecular mass polypeptide bound to DFP (B) is not
immunologically related to VCP. In both panels, proteins were resolved by
SDSIPAGE on 9% gels under reducing conditions. The molecular mass
standards in order of decreasing mass are phosphorylase b, bovine serum

albumin, ovalbumin, and carbonic anhydrase (Bio-Rad).

42

1 2 34 5 1 2 3 4 5
(ill)
41.4

---

46.2

‘5'»-

-g —--. pg --'- .
., «5.0

.. -

-. ~31:

”

 

(A) I (a)

43

the above results, we conclude that VCP is synthesized by the fat body and
internalized by oocytes as a proenzyme, which is then activated in eggs at the
onset of embryogenesis. The activation of VCP is associated with an increase in
its electrophorectic mobility. It is not clear, however, whether this increase in
VCP mobility is due to its proteolytic cleavage or deglycosylation. The mouse
carboxypeptidase (protective protein) is activated as result of cleavage and DFP
binds only to the activated carboxypeptidase subunit that contains the serine
catalytic center (31, 32).

Some of the serine carboxypeptidases with which VCP shares significant
homology, such as human and mouse protective proteins and yeast KEX1
carboxypeptidase, are implicated in proteolytic activation of a number of
enzymes or other biologically active molecules (29-32). The mosquito VCP
could play a similar role by acitvating hydrolytic enzymes that are involved in
degradation of yolk proteins in developing embryos. Alternatively, it could
function as an exopeptidase in sequential degradation of vitellogenin.

The mosquito VCP is different from proteases known to hydrolyze yolk
proteins in insect embryos: an acidic cathepsin B-Iike protease of Drosophila
and a thiol protease or cathepsin L-like protease of Bombyx (8-11). Similar to
mosquito VCP, all these proteases are deposited as proenzymes in yolk bodies
of eggs and activated in embryos. Drosophila cathepsin B-Iike protease is
activated as a result of proteolysis by a serine protease (25 kDa) which is also
present in egg yolk bodies (9). It is not known, however, whether any of these
enzymes are of extraovarian origin or whether they are synthesized by
developing oocytes themselves. The mosquito VCP, therefore, is, to our
knowledge, the first example among oviparous animals of a proteolytic enzyme
shown to be produced by an extraovarian tissue and accumulated by oocytes for

use in embryonic development.

44
ACKNOWLEDGEMENTS

We thank Dr. T.S. Dhadialla and Mr. A. R. Hays for their advice in protein
purification, Dr. H.H. Hagedom for his generous gift of vitellogenin gene probe,
and Drs. T.S. Dhadialla, T.B. Friedman, JR. Miller, and NV. Raikhel for their
critical reading of the manuscript. This work was supported by grants from the
National Institutes of Health and from the Biotechnology Center of Michigan
State University to A.S.R.

45

REFERENCES

1. Kunkel, J.G. and Nordin, J.H. (1985) in Comprehensive Insect Physiology,
Biochmistry and Pharmacology, eds. Kerkut, GA. and Gilbert, L.l.
(Plenum, New York), Vol. 1, pp.83-111.

2. Wyatt, GR. (1988) Can. J. Zool. 66,2600-2610.

3. Dhadialla, T.S. and Raikhel, AS. (1990) J. Biol. Chem. 265, 9924-9933.

4. Kanost, M.R., Kawooya, J.K., Law, J.H., Ryan, R.G., VanHeusden, MC. and
Ziegler, R. (1990) Adv. Insect Physiol. 22, 299-396.

5.Whali, W. (1988) Trends Genet. 4, 227-232.

6. Ferenz, H.-J. (1990) in Molecular Insect Science, eds Hagedom, H.H.,
Hildebrand, J.G., Kidwell, MG. and Law, J.H. (Plenum, New York),
pp131-138.

7. Raikhel, AS. and Dhadialla, T.S. (1992) Annu. Rev. Entomol. 37, 217-251.

8. Medina, M., Leon, P., and Vallejo, CG. (1988) Arch Biochem. Biophys. 263,
355-363.

9. Medina, M. and Vallejo, CG. (1989) Insect Biochem. 19, 687-691.

10. lndrasith, L.S., Sasaki, T. and Yamashita, O. (1988) J Biol. Chem. 263,
1045-1 051 .

11. Kageyama, T. and Takahashi, S. (1990) Eur. J. Biochem. 193, 203-210.
12. Hays, AR. and Raikhel, AS. (1990) Roux's Arch. Dev. Biol. 199, 114-121.

13. Raminani, L.N. and Cupp E.W. (1978) Int. J. Insect Morphol. Embryo]. 7,
273-296.

14. Sambrook, J., F ritsch, E.F., and Maniatis T. (1989) Molecular Cloning: A
Laboratory Manual (Cold Spring Harbor Lab., Cold Spring Harbor, NY),
2nd Ed.

15. Lee, C.C., Wu, X., Gibbs, R.A., Cook, R.G., Muzny, OM. and Caskey, CT
(1 988) Science 239, 1 288-1291 .

16.

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20.

21.

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23.

24.

25.
26.

27.

28.

29.

30.

31.

32.

46

Cho, W.-L., Dhadialla, T.S. and Raikhel, AS. (1991) Insect Biochem. 21,
165-176.

Bose, 8G and Raikhel, AS. (1988) Biochem. Biophys. Res. Commun. 155,
436-442.

Lipman, D.J. and Pearson, W.R. (1985) Science 227, 1435-1441.
Kyte, J. and Doolittle, RF. (1982) J. Mol. Biol. 157, 105-132.
von Heijne, G. (1983) Eur. J. Biochem. 133, 17-21.

Kawooya, J.K, Osir, EC. and Law, J.H. (1986) J. Biol. Chem. 261, 10844-
10849.

Zhu, J., lndrasith, LS. and Yamashita, O. (1986) Biochim. Biohpys. Acta
882, 427-436.

Kulakosky, PC. and Telfer, W.H. (1987) Insect Biochem. 17, 845-858.

Sakai, N., Mori, S., lzumi, S., Haino-Fukishima, K., Ogura, T., Maikawa, H.
and Tomino, S. (1988) Biochem. Biophys. Acta 949, 224-232.

Wang, X.-Y., Cole, KD. and Law, J.H. (1988) J. Biol. Chem. 263, 8851 -8855.
Wang, X.-Y., Cole, KD. and Law, J.H. (1989) Gene 60, 259-268.

Baulcombe, D.C., Backer, RF. and Jarvis, MG. (1987) J. Biol. Chem. 262,
13726-1 3735.

Sorenson, S.B., Svendsen, LB. and Breddam, K (1987) Carlsberg Res.
Commun. 52, 285-295.

Dmochowska, A., Dignard, D., Henning, D., Thomas, D.Y. and Bussey, H.
( 1987) Cell 50, 573-584.

Valls, L.A., Hunter, C.P., Rothman, J.H. and Stevens, TH (1987) Cell 48,
887-897.

Galijart, N.J., Gillemans, M, Harris, A., van der Horst, G.T.J., Verheijen,
F.W., Glajaard, H. and d'Asso, A. (1988) Cell 54,755-764.

Galijart, N.J., Gillemans, N., Meijer, D. and d'Asso, A. (1990) J. Biol. Chem.
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47

33. Hayashi, R., Moore, 8. and Stein, W.H. (1973) J. Biol. Chem. 248, 8366-
8396.

34. Wilson, KP., Liao, D.-l., Bullock, T., Remington, SJ. and Breddam, K.
(1990) J. Mol. Biol. 211, 301-303.

35. Scheiner, OJ and Quigley, JP. (1982) Anal. Biochem. 122, 58-69.

48
CHAPTER 3"

CLONING AND ANALYSIS OF THE LOCUS
FOR MOSQUITO VITELLOGENIC CARBOXYPEPTIDASE

*Originally published in Insect Molecular Biology
Reference: Deitsch, KW. and Raikhel, AS. (1993) Insect Mol. Biol. 2(4), 205-
213.

49

ABSTRACT

Vitellogenic carboxypeptidase is a 53-kDa yolk protein produced by the
fat body of the female mosquito, Aedes aegypti, in response to a blood meal. Its
expression is sex-, stage-, and tissue-specific and is identical to that of the major
yolk protein, vitellogenin. The gene is intronless and two alleles have been
cloned and sequenced, including more than 1.5 kb on both sides of the coding
region. A capsite consensus recently identified as an arthropod initiator was
identified at the start site of transcription. Upstream of this capsite is a 16 bp
imperfect palindrome repeated 4 times showing strong homology to defined
hormone-response elements. In addition, a region that closely resembles the
fat body enhancer and double sex binding site from the Drosophila yolk protein
genes and several potential tat body-specific regulatory protein binding sites

were found.

50

INTRODUCTION

The reproduction of mosquitoes is a subject of intense interest due to the
vectoral capacity of these insects. A key process in egg maturation is
vitellogenesis, the tightly regulated mass production of yolk protein precursors
by the fat body, their secretion into the hemolymph and subsequent uptake by
the developing eggs. This process is unique as it is held in a state-of-arrest in
adult females of anautogenous mosquitoes until a blood meal can be obtained,
at which time the vitellogenic genes are expressed exclusively in the fat body at
extremely high levels, after which expression ceases (for review, see Raikhel,
1992). This provides an excellent model system for the study of tissue-, sex-
and stage-specific gene expression. Analysis of vitellogenic genes should
elucidate the molecular mechanisms behind mosquito egg production and lead
to possible novel methods of mosquito control.

The onset of vitellogenesis in the mosquito Aedes aegypti is marked by
falling titers of juvenile hormone (JH) and rapidly rising titers of 20-
hydroxyecdysone (20E) (Hagedom, 1983, 1985; Raikhel, 1992). The production
of yolk proteins in the fat body is correlated with these hormonal changes,
increasing with rising titers of 20E and ceasing when this steroid hormone's
levels decrease to pre-blood meal levels. In vitro experiments have shown that
physiological doses of 20E are capable of stimulating synthesis of the major yolk
protein, vitellogenin (V9) in previtellogenic fat bodies (Fuchs and Kang, 1981;
Hagedom, 1985). However, in vivo, large doses of 20E are needed to generate
similar levels of V9 synthesis (Fuchs and Kang, 1981; Lea, 1982). In addition,
Vg production is detectable only 1 hr after a blood meal, before 20E titers begin

51

to rise, at both the protein (Raikhel and Lea, 1983) and the mRNA levels
(Racioppi et al., 1986). These results indicate that the regulation of expression
of the vitellogenic genes is complex, and that 205 plays a significant, although
unclear role in this regulation. Little research into the control of expression of
the genes involved in vitellogenesis has been published. Gemmill et al. (1986)
reported the isolation of the mosquito Vg genes and their induction of expression
by 20E. However, the analysis of the genes for this protein is difficult due to
their large size (6.5 kb mRNA) and the fact that they are transcribed from a four
or five member gene family.

Recently, a unique vitellogenic protein was discovered in the mosquito A.
aegypti that is synthesized in a manner indistinguishable from that of V9 (Hays
and Raikhel, 1990). The cDNA encoding this protein was cloned and
sequenced (Cho et al., 1991). The deduced amino acid sequence shares
significant homology with members of the serine carboxypeptidase family. The
protein is synthesized and secreted as a 53 kDa proenzyme by the vitellogenic
fat body. It is internalized by developing oocytes and then stored in the egg until
embryogenesis, at which time it undergoes a reduction in size to the active form
of 48 kDa. The highest levels of the active enzyme are found at
midembryogenesis, after which the protein disappears. These results show that
an enzyme involved in embryogenesis is produced by an extraovarian tissue,
stored in eggs and activated later in development; a phenomenon not previously
known for oviparious animals. Reflecting these unique characteristics, the
protein was named vitellogenic carboxypeptidase (VCP).

Here we describe the analysis of the locus for VCP, a gene that shows
identical temporal, tissue and levels of expression to vitellogenin, but that is
single copy, intronless and much smaller. Two alleles for this gene have been

cloned and sequenced, including ~1.5 kb on both sides of the coding region.

52

Analysis of the regulatory regions provides a starting point for molecular
characterization of the hormone responsiveness of the genes involved in

vitellogenesis.

53
RESULTS

Cloning and Sequencing of VCP Genomic Fragments

Previously, the cDNA encoding VCP had been cloned and sequenced
(Cho et al., 1991). Using an 840 bp fragment of this sequence as a probe,
several genomic clones were obtained by screening a A Fix ll Aedes aegypti
genomic library. Restriction mapping indicated that these DNA fragments could
be classified into two groups, arbitrarily named VCP1 and VCP2 (Fig. 1).
Genomic blots probed with the VCP cDNA showed bands corresponding to both
VCP1 and 2. Because preparations of genomic DNA are typically extracted from
large numbers of mosquitoes which are not genetically homogeneous, it was not
clear whether VCP1 and VCP2 represented two copies of the VCP gene in the
A. aegypti haploid genome, or simply two separate alleles existing within the
population. To differentiate between these two possibilities, genomic blots of
DNA extracted from individual mosquitoes were probed with the VCP cDNA.
The resulting autoradiograph showed clearly that VCP1 and VCP2 are two
single copy alleles and that homozygotes for both alleles as well as
heterozygotes exist (Fig. 2). The results were identical regardless of whether
DNA was extracted from male or female individuals. The intensity of
hybridization was similar when comparing lanes of male DNA to DNA extracted
from females or female fat bodies, indicating that specific amplification was
unlikely. To confirm the copy number determination, 0.5, 1.0 and 2.0 copy
equivalents of the VCP cDNA were blotted next to 10 ug of genomic DNA and
the intensity of the hybridization with the VCP probe compared. This method
also indicated a single copy gene (data not shown). Recently, using restriction

fragment length polymorphisms, Severson et al. (1993) have mapped VCP to a

Fig. 1: Restriction maps of the two alleles of VCP. The open box represents
the coding region. Hatched box represents the inserted DNA unique to VCP2.
The striped white bars show the downstream non-homologous region of the two
alleles. The length of each restriction fragment is indicated in base pairs above
the map. The approximate site of several putative transcriptional control

elements is also indicated.

55

3 one

ICPZ
19 two
above

nnlrol

fimcom H m.

”:0me Dv— m .N..(

 

. we a w:fl S
SEES c. .e b

.5382

 

~mo>

Fig. 2: Genomic blots of DNA from individual mosquitoes. (A) Diagram
showing the hybridizating fragments distinct to the two alleles. (B) Blots showing
individuals homozygous for VCP2 (lane 1), VCP1 (lane 2) and the corresponding

heterozygotes showing both alleles (lane 3).

57

 

 

 

VCP1

 

 

 

 

 

l 4120 hp J
EcoKfi genomic frag 70R]
1, ' “7 I; ' , H I coding region I ' ' . l
-1066 +1 .1512 .3054

 

VCPZ

2391 00 1445 bp

Ecoll\1 genomc frag genomc lrag 70m

 

 

 

Ecoer 1038

 

_, » I coding region r ’ ‘ . J
-1353 01 91513 92483

‘15 .840

23.1—

9.4—
6.6—

4.4—

2.3—
2.0—

VCP l (4120”)

WP 2 (2391 hp)

58

single site in the mosquito genome between the marker LF181 and the y-
aminobutyric receptor subunit A receptor gene.

The differences in the restriction maps of the two alleles for VCP are
much greater than one would anticipate for two alleles found within a single
inbred population. When a second gene was cloned from this population of
mosquitoes, that encoding lysosomal aspartic protease, a number of distinctly
different alleles were also found (Dittmer et al., unpublished). This lab strain of
Aedes aegypti was founded approximately 20 years ago, and the origin of the
founding mosquitoes is not known. It is possible that individuals from distinctly
different populations were brought together in the initial founding of the colony.
This could have led to the rather stark differences in the alleles shown here for
VCP.

1,716 bp upstream and 1,542 bp downstream of the coding region in
VCP1 and 1,353 bp upstream and 970 bp downstream of the coding region in
VCP2 were sequenced. The coding regions in both VCP1 and VCP2 included
no introns and encoded 100% identical polypeptides. The nucleotide sequence
of the two alleles is also highly conserved (~97% identity) with the exception of
two unique, non-homologous areas (Fig. 1). Sequence analysis has shown that
614 bp upstream of the start site of VCP2 a ~2.1 kb fragment exists which is
not found in VCP1. This represents either an insertion in VCP2 or a deletion in
VCP1. Also, cross-hybridization analysis of the two alleles indicates that
downstream of the open reading frames there is a ~3 kb region with
considerably less homology. The complete nucleotide sequence of VCP1 is
shown in Fig. 3, including a putative TATA box and ribosomal binding site as
defined by Hagenbuchle et al. (1978).

59

Fig. 3: Complete genomic sequence of VCP1: Inverted arrows represent the
putative hormone response elements (-561 to -499); the single black bar shows
the arthropod initiator (+2 to +6); the TATA sequence is boxed (-22 to -29); +1
indicates the start site of transcription; capital letters show the translated portion
of the gene; double underline is the putative ribosomal binding site (+22 to +25);
single underline shows the polyadenylation signal (+1389 to +1394); r-—-r

identifies the region of similarity with the Drosophila yolk protein fat body
enhancer (+1627 to +1657); - - - - shows the consensus double sex binding
sequence; --- identifies sequences with similarity to the BBF-2 binding site; III

shows sequences with similarity to CREB/ATF binding sites.

-1702

-1627

-1552

-1477

~1402

-1327

-1252

—1177

~1102

-1027

-952

~877

-802

-727

-652

-S77

-502

-427

-352

-277

-202

-127

-51

+25

+100

+175

+250

+325

+400

+475

+550
+625

ccaatcctggtgcaggtgatccagtatttgccaccccaatttttaatacaaaaacaaattttgtgaccagttctc
atatttttccttctaaaaa att aaa ctttcatttaac tctt acagtattcagaagtctattaataaaaa
ccoco-lgg g g I/f/g/l/ g
atcataattataaaacatattttttccttagaatgacaaaaactaatacgtctggcctatgttcaagataacaac
atgccagattccatagttgaaacccgaaatcaagaagaatcttttgtataattcatgaagaaatctattgaagat
ttcttgttgaaatccatgaaaagttttttcgatgattgtctgtaaataatatttaagaaatacctgaaacatttg
tt aa at tca taatactcaattct a acatc ta aaaacattct a aaacctcc a aaaaact
9 33/I3/xyg 99 9 9 99 99 9 9 99
aaaaatttgctgttttatat attat attttattttttatctcgtttctggc ttaagtcccaact at ca
9 g g IVz/glz/ 9 9 g
agcctacttttcagctttttgcatctttttgcatgcgtatatcgtgtggcaggtacgatggtactatatgccttg
tggataactgtgcgttaactgctacggctatctaggcccccatataactatattatttagagsggaasctttgga
ataatactgagaagagtccctgtaatatggagggaggattttcgaaagcgatttctgtaaattttafgaagctcc
tgtatttcggacgaaaactgcttagcaattccccaattctgaaagaacgacttattgaaacgtgtgaaaatatta
ttactatttctgggcttgtattgtgttcctagaagaatcccagatgtttatgatcttattcttgaagaaatgcac
ttaggactttaacccctcaccggcagctatacataaatgtacataaaacactataatttttttgattttcaacaa

tttttttaccattgttccgcaagttcccaaatacttttctgtttccagaacggttctatcccattaggccgaatg

 

EggtsaggctgaaSgEEgttaggccgaaggccattagaccgaaagagtcgttaggccgaaagagtcgttagaccg
—>--+

 

aaaatggcagaaagaacagcctaaatatgcttaaaagaaggaaaacacactgatgaatgtgttagccattEggs;
Seacgaccctttcggct599c 9a Sgttctgcctaacgacctgcacccaatagaagagttttttgagaacgtttaa
aaatgttgcaaaaatattgtcaaacaaaaatgttatagcgaccacattatattttgtggtaaaaacaaataagct

gcctgtagcaaatttattgtgcgatgtaagacaaaacgttagatctgagatgatcacaaaaaagtacagtaa2Ea

aaaattacatatcccctacagcaaaacacgctccatctcctagaaaaaatcctagctacaccaatgattgtcact

Oll-

tggatcatgtttttctatactgttcattgacttcgatcgttgtgaggagcgatcattatcgcatcaagatcagca
0...... I

- +

agatttcttcaatcggcttato- ggaccatatgagtat:tacgatlzgccagtggtcttttgtggccgjht
ggttgtaaagtgtaatcgaacaagcATGGTGBARTTCCATTTACTAGTGCTGATTGCGTTCACCTGCTATACATG
CAGCGACOCAACCTTATGGAATCCGTACAAGAAGCTTATGAGAGGATCGGCGTCTCCTCCTCGTCCAGGTGAAAG
TGGTCAACCTTTGTTCCTGACTCCACTGTTGCACGATCGCAAAATTGAAGAAGCTCGCAACAAAGCCCGTGTCAA
CCATCCCATCTTGAGCTCACTGGAAAGCTACTCCCGTTTTATGACCGTTCATGCCAAGCACAACTCCAATTTGTT
CTTCTGGTATGTCCCAGCTAAGAACAACCGTGAACABCCGCCCATTCTTCTTTGGTTGCAAGGAGGTCCAGGTGC
GTCATCGCTGTTCCGAATGTTCGAACAAAATGGACCGTTCCATATTCACAGGAACAAATCAGTCAAGCAACGTCA
ATACTCCTGGCATCAGAACCATCACATGATCTACATCGATAATCCAGTTGGAACGCGATTCAGTTTCACCGATAG
CGATGAAGGTTACTCCACCAACGAAGAACACGTGGCTGAAAATCTGATGAAATTCATTCAACAATTTTTCGTGCT
CTTCCCCAATCTGTTGAAGCATCCATTCTACATCTCCGGTGAATCTTATCGTGGAAAGTTTGTTCCTGCCTTCGG

+700

+775

+850

+925

+1000
+1075
+1150
+1225
+1200
+1275
+1350
+1425
+1500
+1575
+1650
+1725
+1800
+1875
+1950
+2025
+2100
+2175
+2250
+2325
+2400
+2475
+2550
+2625
+2700
+2775

+2850

+2925

61

TTATGCCATCCACAACTCCCAAAGCCAACCTAAAATCAATCTGCAACGATTGGCCATTCGTGATGCCTACACTCA
TCCCCTGAACCAACTTAACTACGGAGAATACCTCTATCAGCTCCCCCTGATCCATTTGAACGGAAGAAACAAGTT
CGACGAGGATACCGCTCCTCCCATCGCCTGTGCCGAACGTAACGACATGAACTCTGCCAATCGCCTTATCCAAGG
TCTGTTCGATGGACTCGATGCCCAGCAATCGTACTTCAACAAGGTCACCGGATTCTCCTCCTACTACAATTTCAT
CAAGGCAGACGAGCAGAGCAAACACGATAGTGTCCTGATGGAGTTCCTCAGCAACCCCGAGGTACOTRAGGGCAT
CCACGTTGGTGAACTCCCGTTCCACGACTCTGACCGTCACAACAACGTCGCGCAAATCCTCTCCGAAGACACTCT
CGACACCGTGGCTCCATGGGTCACCAAGCTGCTCTCGCACTACCCCGTGCTCTTCTACAACGGTCAGTTGGACAT
CATCTCCGCCTACCCGATCACGGTCGACTTCCTGATGAAGATCCCTTTCCATGGCCATAGCGAGTACAAGCGCGC
CAATCGTGAGATCTACCGCGTCGATGGTGAAATCCCCGGGTACAACAAGCGGGCACGCCGCCTGCAGGAGGTCCT
CATCAGAAACCCCGGACACATCCTCCCGCGGGATCACCCGRAGTGGGCATTCGACATCATCACCTCGTTCACTCA
CAAAAACTACTTGTGAGGGAATGGCAATGATTTGGATGAATAAACCATTGAGCTCTAATTAAAGAAAgcgttcct

atctttatttgtagaatatcagttttagcgactcctttcattcttctgacattgtatcccaattgaggcaaagac

tacactctatcaaaatattctgccttcgtgagcacttccactgttaggasgEgagatttttttttctgttggagg

 

cattgcacagtgggacggattgaggttttaggaggaaagatggaactcacgcgsgcaagggtgattttacgtaaa

aataatgttcEacaaagttgtttctatcataaaaacaatttttttggtcggaacgaaaattagggtggccctatg

ttaaaaaagataaccatcaaaacttttttaatttacggaatattgatatagtttgttcgacaaagstgaagatca
aaaaatttcaagctgatttgataaaaaagttttttcctagctcaaaaattgaccgttttagagcatttttcgcta
ttgatgtagggtggcccatcaaaaattgtttttttttgtgtttaatttttattatttcaagtttcttagcaaaat
tgtcttcccattactttcaagactaattgaaacgcaaacataaaagatatagctaatggaaacaaatagatacaa
gaatcgttttgtaatgaaaatttaatttacttccttggacataaagtatcatcgatactaatacatacgatatac
gaafgcaaaatgaaaaatatggcaaagaaagctctcagttaataactgtggaagtgttcctaacaacaataaaaa
aaggaatattgagacaagssttttcgatgaaaagttaaagactttttgateaccaaaaaaaaaacatgtttatat
gtttgtatacacacttagaaaatatcaccgactccggtaatttttttaccgaaatataaaccgctgagcgctcag
taatgctttcggtaaagttaaggattaccgaacaatctgtaattgcaatcgactcgcagctgtcaaaaaagggga
ascggctcgtgaattattaccggacgtattgtaaaataaactaccgaacgcattattcgta5caacaggcagtcg
gaatagaaaaggatgatcaeaacaaaggaaaaaccatattgatagcaaatgttcatcgatttatttcgcattttt
gttttgaagccgtttgaagatgaactgcttgtctttttcatgaagatggaagatgataccacccccagggggggg

I
,ccaacaactcgaacgcgcgcttcaaatcttgggctacaatcctggctcatttgcagctgttgaccccttttacc
000.0000.

accaccaccaccga$$ssg$99tatgtgtttatcctcgtttatgtgacgattctatccggaaacgtaattgggca

gttggaaactacaaggaaaatattttattcaacttttaaaaatagttttttttataaaattccccaggtcttact
0.0000000. - '

tacggaaaagtagttccagacgctcatcctgctgtcgtagtctctattttttttttctcaaaaaacagtgtttat

ggtcacaatcacagaaattaaacggattgaatt

62

Identification of the "Arthropod Initiator" and Capping Site

Examination of VCP1 and VCP2 also showed the sequence TCAGT,
recently identified as an arthropod initiator, 24 bp downstream of the putative
TATA box, indicating that this sequence may also be located at or near the
transcriptional start site in this gene (Fig. 3). The capping sites of the Drosophila
yolk protein genes 1 and 2 show a sequence very similar to this Alnr (Hung et
al., 1983) (A; Fig. 4). To confirm that the true start site is localized in this region,
a primer extension experiment was done, showing that +1 is in fact immediately

upstream of this consensus capsite sequence (B; Fig. 4).

Putative Hormone Response Elements

Inspection of the untranscribed regions surrounding the VCP gene
revealed the existence of a 16 bp imperfect palindrome repeated 4 times (Fig.
3). All four repeated elements are located in close proximity to one another
upstream of the VCP start site and show strong similarity to previously described
hormone response elements (Fig. 5). The arms of the palindromes are 8 bp long

and extend directly to the center of the element, leaving no spacer.

Homology to Fat Body Enhancers and Protein Binding Sites from
Drosophila

Several regulatory elements and protein binding sites have been
identified in Drosophila that confer fat body specific expression to the genes
under their control. These include the 127 bp fat body enhancer and doublesex
(DSX) binding site originally identified between the YP1 and YP2 genes (Burtis
et al., 1991;Garabedien et al., 1986), several recently identified fat body
enhancers also found between YP1 and YP2 (Abrahamsen et al., 1993) and the

63

Fig 4. Identification of the transcription start site for VCP as well as a
putative Arthropod initiator. (A) Comparison of capsites from two alleles of
VCP with those from the DroSOphila yolk protein genes 1 and 2 and the
arthropod initiator consensus sequence. (B) Primer extension analysis showing
the start site of transcription. The extension product is run next to two
sequencing ladders for markers; one from VCP genomic DNA and the second

from an M13 standard. (C) Diagramatic representation of the extension reaction.

A.

Consensus Capsites: Putative Arthropod Initiators

Consensus: T C A G T

VCP1 I CCAGTGG
VCPZ I CCAGTTG
Dros.YP2: CCAGTTC
Dros.YPl: CCAATCG

B.

 

Gznomic DNA Extension :13mp186
product
T
T
A
c
3‘
-1' 91 bp
G
C
C
A
G/
T
Arthrop 0d
lnltlator
( 3 +1
' TTACGATGCCAGT

 

l—

<..

\./l

 

30 mcr pn'mcr

L____
91 nt

65

Fig. 5: Response element comparisons. Comparison of the putative hormone
receptor binding sites from VCP with the consensus EcRE from Drosophila
(Cherbas and Cherbas, 1993).

G
Consensus EcRE: R G TT C A

W?

TCGTTAGG
TCGTTAGG
TCGTTAGA

CCATTAGG

n

C
TGAACY
CCGAAAGA

CCGAAGGC
CCGAAAAT

CCGAATGC

67

binding sites for the box B-binding factor-2, CREB (cAMP responsive
transcriptional enhancer binding protein) and ATF (activating transcription
factor) defined for fat body specific expression of the alcohol dehydrogenase
gene (Abel et al., 1992; Hai et al., 1989; Hoeffler et al., 1988). Using
GOG/Pasta, these enhancer regions and consensus sequences were compared
to the untranscribed portions of the VCP gene.

In both alleles of VCP, a sequence downstream of the coding region was
identified as having similarity to the 127 bp fat body enhancer identified by
Garabedien et al. (1986) (Fig. 6). Several short stretches of homology were also
identified when the fat body specific enhancers described by Abrahamsen et al.
(1993) were used for comparison. These areas tended to be AT rich and less
similar than the previous fat body enhancer, indicating they maybe of less
significance. Six potential binding sites for BBF-2 and 12 sites matching the
CREB/ATF binding sequences were identified scattered throughout the VCP
untranscribed DNA (Fig. 3).

Fig. 6: Fat body enhancer comparison. Region of similarity identified through
computer comparison of the fat body enhancer from Drosophila yolk proteins 1
and 2 (Garabedien et al., 1986) and the untranscribed region of both alleles of
VCP. The consensus binding sequence for double sex is identified (DSX)

(Burtis et al., 1991).

69

Possible Fat Body Enhancers

VCP1Clifil’l”i‘i‘lliTT’lCGlAl‘l‘ll‘l’i’ilG

DROS: CTAGAAA- GTGATTACAAATI'TI'PIIAAATAATC

vcpz; CTACAAA GTTTT TATAC TTACCACCGCT

‘\ /‘J

DSX Binding Site

eins 1
es of

DSX)

70
DISCUSSION

Using the cDNA for VCP as a probe to screen an Aedes aegypti genomic
library, genomic clones representing two alleles for the VCP locus have been
identified, sequenced and analyzed. The sequences show several elements
indicative of the hormone responsiveness and sex-, stage- and tissue-specific
expression of this gene. These include 4 putative hormone receptor binding
sites, an arthropod initiator, a region of similarity to a DroSOphila fat body
enhancer, and several potential regulatory protein response elements. The
exact start site of transcription was mapped to within two base pairs of this
initiator.

Cherbas and Cherbas (1993) theorized that the capsite consensus found
in the promoters of many arthropod genes, termed the "arthropod initiator"
(Alnr), is a functional homologue to the TdT family of mammalian initiators.
Experiments done with the control regions of the 20E inducible gene Eip 28I29
and the ecdysone response element (EcRE) of hsp27 in Drosophila indicated
that hormone response elements and the Alnr work in conjunction with one
another, and are dependent on each other to induce expression (Cherbas and
Cherbas, 1993). The expression of VCP is likely also dependent on the binding
of a honnonelreceptor complex to several putative binding sites upstream of the
capsite. The function of these binding sites may depend on the presence of the
Alnr in much the same way as the 20E binding sites are dependent on this
sequence in Eip28l29 hormone induced expression.

The promoter and enhancer regions of steroid hormone responsive
genes defined in other organisms show many conserved motifs. In general,
these regulatory regions contain hormone response elements, which tend to be

imperfect palindromes 12-16 bp in length, often present in multiple copies,

71

called hormone response units (for review, see Martinez and Wahli, 1991). The
structural features critical to the activity of EcREs in Drosophila have now been
rigorously examined (Riddihough and Pelham, 1987; Cherbas et al., 1991).
Recently, Antoniewski et al., (1993) have shown through mutational analysis
using the hsp 27 and Fbp genes that the integrity of each half site of the
palindrome as well as the 1 bp spacing is critical for binding of the ecdysone
receptor (EcR). They offer a revised version of a previously proposed EcRE
consensus sequence and state that variations in this sequence may result in
binding sites having different affinities for the receptor.

The repeated imperfect palindrome found upstream of the gene for VCP
resembles the described EcRE from Drosophila and also strongly resembles a
retinoic acid response element (de The at al., 1990) (Fig. 5). These repeated
palindromes may represent a hormone response unit responsible for the tightly
regulated, high levels of expression of this gene and may shed light on the
expression of several vitellogenic gene products. The potential binding sites are
similar to but not exactly like the consensus EcRE described by Antoniewski et
al. (1993). In particular, the palindromes found in the VCP upstream region have
no spacer, while the responsiveness of the EcRE from Drosophila was shown to
be dependent upon a 1 bp spacer. These differences, combined with the recent
research by Cho et al. (1993) showing that EcR in mosquito is expressed only at
very low levels in the vitellogenic fat body indicates that this gene may be
responding to a cellular signal other than 20E. The Ashbumer model explains
how 20E in complex with its receptor directly causes the expression of certain
early response genes, many of which encode transcriptional activators that act
on genes downstream in the pathway (Ashbumer et al., 1974). It is possible that

the VCP gene of mosquito is regulated by a similar cascade, and that the

72

transcriptional activator binding upstream of VCP may be a protein further
downstream in the 20E response hierarchy.

Due to a lack of germline transformation or vitellogenic cell lines which
would enable us to investigate transcriptional regulation by specific promoter
and enhancer regions directly, computer aided comparison of sequenced
regulatory regions was used to identify possible control elements within the VCP
gene. This comparison identified several possible control elements scattered
throughout the untranscribed region, including several in close proximity to one
another. Computer comparisons should be viewed with caution. Abrahamsen et
al. (1993) showed how searches for protein binding sites can identify consensus
sequences in nearly every sequence compared, including those not regulated by
the binding proteins. However, these searches can provide useful information
for further analysis including gel retardation and footprinting assays.

The vitellogenic genes of the female mosquito are expressed in a sex-,
stage- and tissue-specific manner. The gene for VCP is a small, concise gene
representative of this important developmental cycle. It is hoped that the
identification of possible hormone response elements as well as potential
regulatory protein binding sites will prove useful in determining the molecular

mechanisms controlling this expression.

73
EXPERIMENTAL PROCEDURES

Animals

Mosquitoes (UGAL strain of Aedes aegyptr) were reared at 27° C and
85% relative humidity. Larvae were fed on a standard diet (Lea, 1964). Adults
were fed 10% sucrose by wick continuously. Three to five days after eclosion

adult females were allowed to feed on white rats to initiate vitellogenesis.

Genomic Library Construction and Screening

Genomic DNA was extracted from approximately 0.5 g of mosquito whole
bodies using a standard DNA extraction procedure (Ausubel, et al, 1989). The
tissue was frozen in liquid N and ground to a fine powder using a mortar and
pestle. It was then suspended in 5 ml of digestion buffer (100 mM NaCI, 10 mM
Tris-Cl pH 8.0, 25 mM EDTA, 0.5 % SDS, 0.1 mglml fresh proteinase K) and
incubated at 50° C overnight. The solution was then extracted gently with 1 vol.
phenol/chloroformlisoamyl alcohol (25:24z1) and centrifuged at 10k rpm
(Beckman J-20 rotor) for 10 min. The aqueous phase was saved and the
extraction repeated until no interphase was present. The DNA was then
precipitated with the addition of 0.5 vol. 7.5 M NH4-acetate and 2 vol. ethanol.
This was spun down at 5k rpm for 2 min., rinsed with 70% ethanol and allowed
to air dry. The resulting DNA pellet was gently resuspended in 1 ml of TE
(10mM Tris-Cl, 1mM EDTA, pH 8.0). This DNA was sent to the Stratagene Corp. .
and a 1.le ll genomic library constructed. This library was screened according
to the manufacturers instructions using an 840 bp [a-32P] random primer labeled
VCP cDNA fragment (Cho, et al., 1991). Resulting positive clones were verified
through Southern analysis, restriction mapped and selected DNA fragments
subcloned into pUC 118 and pUC 119 for sequencing.

74

DNA Sequencing

Subcloned genomic fragments were sequenced in both directions using
the Sequenase (USB Corp.) sequencing kit and the manufacturer's protocols.
Unidirectional deletions were generated using the method of Dale (1985) with
M13 derived single stranded DNA The resulting sequences were analyzed
using Dotplot, Fasta, and Compare programs (University of Wisconsin Genetics

Computer Group software) and through visual inspection.

Genomic Blots

Genomic DNA was isolated as previously described, digested to
completion with restriction enzymes, and fractionated using 0.8% agarose gel
electrophoresis. The DNA was then transferred to nitrocellulose membrane
using the method of Southern (1975) and probed under high stringency
conditions (5X SSC, 50 mM phosphate buffer, 50 % formamide, 5X Denhardt's,
0.01 % SDS, 200 uglul salmon sperm DNA). The resulting blots were washed in
2X SSC, 0.1% SDS at room temp twice, in the same buffer at 60° C once, and in
0.2x SSC, 0.1% SDS at 60° C once. The blots were allowed to air dry and were
used to expose film at -70° C using an intensifying screen. Genomic DNA from
individual mosquitoes was isolated by homogenizing single mosquitoes in 400 pl
digestion buffer, followed by the previously mentioned protocol scaled
accordingly.

Primer Extension

The primer extension experiment was done following the methods
described by Sambrook et al. (1989) and Ausubel et al. (1989). 150 ng of a 30
bp synthetic primer derived from the VCP cDNA sequence was and labeled with

75

[ii-3’43] ATP by T4 polynucleotide kinase. This was hybridized to 10 ug of total
RNA extracted from vitellogenic fat bodies isolated 20 - 24 hr after a blood meal.
AMV reverse transcriptase was then used to reverse transcribe labeled DNA
from the RNA template. This was extracted with phenol/chloroformlisoamyl
alcohol (2522421) and precipitated with 3 vol. ethanol in a dry ice/ethanol bath.
The DNA was spun down in a microcentrifuge at 4° C, rinsed with 70% ethanol
and allowed to air dry. The dry pellet was taken up in 4 ul H20 and 6 ul
fonnamide loading buffer (80% formamide, 10mM EDTA pH 8.0, 1 mglml xylene
cyanol FF, 1mglml bromophenol blue). 5 ul of this was run on a 6%
polyacrylamide denaturing sequencing gel. A sequencing reaction done with
single stranded DNA derived from a VCP genomic clone using the same
synthetic primer was used as a marker. An additional marker was derived by
sequencing M13mp18 single stranded DNA The resulting autoradiograph

shows the start site of VCP mRNA transcription.

Computer Analysis

The VCP untranscribed regions were compared with the untranscribed
regions of the Drosophila yolk protein genes using the Fasta program (University
of Wisconsin Genetics Computer Group software). To identify possible DSX
binding sites, 7 bp homology to either the suggested consensus sequence
(CTACAAAGT) or to the sequence CTACAATGT which was shown to exhibit the
strongest affinity for the DSX protein was searched for. Similar searches were
done for the consensus binding sites of CREB/ATF (TGACGTCA) and BBF-2
(TAICNACGTANTIGC).

76
ACKNOWLEDGEMENTS

We would like to thank Dr. Wen-long Cho for his advice throughout the
course of this study. We also thank Dr.Wen-long Cho, Jeng-Shong Chen and
Neal Dittmer for their critical reading of the manuscript. This research was

supported by grant Al-24716 from the National Institutes of Health to ASR.

77
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transcriptional activator binds to both fat body- and liver-specific
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Abrahamsen, N., Martinez, A., Kjaer, T., Sondergaard, L. and Bownes, M.
(1993) Cis-regulatory sequences leading to female-specific expression of
yolk protein genes 1 and 2 in the fat body of Drosophila melangaster.
Mol. Gen. Genet. 237: 41-48.

Antoniewski 0., Laval M. and Lepesant J. (1993) Structural features critical to
the activity of an ecdysone receptor. Insect Biochem. Mol. Biol. 23: 105-
1 14.

Ashbumer M., Chihara C., Meltzer P. and Richards, G. (1974) Temporal control
of puffing activity in polytene chromosomes. Cold Spring Harbor Symp.
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Ausubel F. M., Brent R., Kingston R. E., Moore D. D., Seidman J. G., Smith, J. A.
and Struhl, K. (1989) Current Protocols in Molecular Biology, 2.2.1-
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Burtis, K. C., Coschigano, K T., Baker, B. S. and Wensink, P. C. (1991) The
doublesex proteins of Drosophila melanogaster bind directly to a sex-
specific yolk protein gene enhancer. EMBO 10:2577-2582.

Cherbas L. and Cherbas P. (1993) The arthropod initiator: the capsite
consensus plays an important role in transcription. Insect Biochem. Mol.
Biol. 23: 81 -90.

Cherbas, L., Lee, K and Cherbas, P. (1991) Identification of ecdysone
response elements by analysis of the Eip28l29 gene. Genes Dev. 5:
120-1 31 .

Cho W., Deitsch K W. and Raikhel A. S. (1991) An extraovarian protein
accumulated in mosquito oocytes is a carboxypeptidase activated in
embryos. Proc. Natl. Acad. Sci. USA. 88: 10821-10824.

Cho W., Kapitskaya M. Z. and Raikhel A. S. (1993) Mosquito ecdysteroid
receptor: analysis of the cDNA and transcription during vitellogenesis.
Proc. Natl. Acad. Sci. USA. In Press.

78

Dale R. M. K, McClure B. A. and Houchins J. P. (1985) A rapid single-stranded
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de The H., Vivanco-Ruiz M.M., Tiollais P., Stunnenberg H. and Dejean A.
(1990) Identification of a retinoic acid responsive element in the retinoic
acid receptor B gene. Nature 343: 177-180.

Fuchs, M. S. and Kang, S-H. (1981) Ecdysone and mosquito vitellogenesis: a
critical appraisal. Insect Biochem. 11: 627-633.

Garabedien M. J., Shepherd B. M. and Wensink PC. (1986) A tissue-specific
transcription enhancer from the Drosophila yolk protein 1 gene. Cell 45:
859-867.

Gemmill R. M., Hamblin M., Glaser R. L., Racioppi J. V., Marx J. L., White B. N.,
Calvo J. M., Wolfner M. F. and Hagedom H. H. (1986) Isolation of
mosquito vitellogenin genes and induction of expression by 20-
hydroxyecdysone. Insect Biochem. 16:761-774.

Hagedom, H. H. (1983) The role of ecdysteroids in the adult insect, in Downer,
R. G. H. and Laufer, H. (eds): Endochrinology of Insects, Alan R. Liss,
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Hagedom, H. H. (1985) The role of ecdysteroids in reproduction, in Kerkut, G.
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Hai, T., Liu, F., Coukos, W. and Green, M. (1989) Transcription factor ATF
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2090.

Hays A. R. and Raikhel AS. (1990) A novel protein produced by the vitellogenic
fat body and accumulated in mosquito oocytes. Roux's Arch. Dev. Biol.
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Hoeffler, J. P., Meyer, T. E., Yun, Y., Jameson, J. L. and Habener, J. F. (1988)
Cyclic AMP-responsive DNA-binding proteins: structure based on cloned
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79

Hung M. and Wensink PC. (1983) Sequence and structure conservation in
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Lea, A. (1964) Studies on the dietary and endocrine regulation of autogenous
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Martinez E. and Wahli W. (1991) Characterization of hormone response
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Racioppi J.V., Gemmill R. M., Kogan P. H., Calvo J. M., and Hagedom H. H.
(1986) Expression and regulation of vitellogenic messenger RNA in the
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Raikhel A. (1992) Vitellogenesis in mosquitos. Advances in disease vector
research. 9, 1-39. Springer-Verlag, New York.

Raikhel A. S. and Lea A. O. (1983) Previtellogenic development and
vitellogenin synthesis in the fat body of a mosquito: an ultrastructural and
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Riddihough, G. and Pelham, H. R. B. (1987) An ecdysone response element in
the Drosophila hsp27 promoter. EMBO 6: 3729-3734.

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80

CHAPTER 4*
INDIRECT CONTROL OF YOLK PROTEIN GENES BY 20-
HYDROXYECDYSONE IN THE FAT BODY OF THE MOSQUITO, AEDES
AEGYPTI

*Submitted for publication to Insect Biochemistry and Molecular Biology
Reference: Deitsch, KW., Chen, J.-S., and Raikhel, AS. (1994) Insect Biochem.
Mol. Biol., Submitted.

81
ABSTRACT

In response to a blood meal, the fat body of the female mosquito, Aedes
aegypti, begins massive production of several yolk proteins which are
subsequently stored in the developing oocytes. Although 20-hydroxyecdysone
(20E) is important for initiation and maintenance of expression of the yolk protein
genes, the exact nature of 20E action has not been clearly defined. A primary
question is whether this hormone directly stimulates yolk protein mRNA
transcription or if it acts indirectly through a hormone response cascade. We
have demonstrated that 10'4 M cycloheximide (Chx) reversibly inhibits >98% of
protein synthesis in in vitro fat body primary culture. 10'5 M 20E stimulates high
levels of yolk protein gene transcription, but initiation of this transcription is
eliminated by Chx. Thus, our results indicate that protein synthesis is required
in response to 20E before transcription of the yolk protein genes is initiated. We
therefore conclude that the action of 20E is indirect.

82
INTRODUCTION

In the anautogenous mosquito, Aedes aegypti, vitellogenesis is initiated
by a blood meal. The fat body transcribes large quantities of yolk protein mRNA
and massive amounts of these proteins are produced and transported to the
developing oocytes. The titer of 20-hydroxyecdysone (20E) is tightly correlated
with this cycle, rising with yolk protein production and falling when vitellogenesis
ceases (Hagedom, 1983, 1985; Raikhel, 1992). Initial experiments on the role
of 20E in yolk protein regulation have been inconclusive. In vitro, physiological
doses of 20E can stimulate synthesis of the major yolk protein, vitellogenin (V9)
in previtellogenic fat bodies (Fuchs and Kang, 1981; Hagedom, 1985).
However, large doses of 20E are needed to generate similar levels of V9
production in vivo (Fuchs and Kang, 1981; Lea, 1982). In addition, induction of
V9 synthesis is detectable at both the protein (Raikhel and Lea, 1983) and the
mRNA levels (Racioppi et al, 1986) only 1 hr after a blood meal, before 20E
titers begin to rise. These results show how complex the regulation of
vitellogenic gene expression is, and that 20E plays a significant, although
unclear role in this regulation.

A second yolk protein has been described called vitellogenic
carboxypeptidase (VCP) (Hays and Raikhel, 1990; Cho et al., 1991). In vivo and
in vitro experiments indicate that this protein is synthesized in the same tissue-,
stage- and sex-specific manner as Vg. Recently, cDNAs encoding both V9 and
VCP have been cloned and sequenced (Cho et al., 1991; Chen et al., 1994),
providing molecular probes for investigating the role of 20E in the control of
these two genes.

Ashbumer et al. (1974) proposed a model for the mode of action of 20E in
Drosophila. They hypothesized that 20E, complexed with its receptor, directly

83

regulates two classes of genes: a small class of early genes that are transcribed
when bound by this complex and a large class of late genes that are repressed.
The early genes encode transcription factors that serve two functions. They
repress early gene transcription and they induce late gene transcription. Recent
research at the molecular level has revealed many genes whose regulation is
consistent with this model (Segraves and Hogness, 1990; Burtis et al., 1990;
Koelle et al., 1991). Thus, 205 is thought to operate through a cascade or
hierarchy of genes initially induced by the 20E/receptor complex and ultimately
leading to expression of numerous downstream target genes. It is important to
determine whether 20E and its receptor act directly to initiate yolk protein gene
transcription in mosquito or if they act through a hierarchy of upstream
transcriptional activators in a response cascade. The results presented here
support the latter hypothesis, indicating that the Ashbumer model also likely

applies to the genes involved in mosquito vitellogenesis.

84
MATERIALS AND METHODS

Insects

Mosquitoes were reared at 27° C and 85% relative humidity in a 16l8
(light/dark) cycle. Larvae were fed on a standard diet (Lea, 1964) and adults
were fed 10% sucrose continuously by wick. Three to 5 days after eclosion,
adult females were allowed to feed on white rats to initiate vitellogenesis. All
dissections were performed in Aedes physiological saline (Hagedom et al.,
1977) at room temperature. For culture, isolated abdominal cuticle with
adhering fat bodies were used immediately after dissection. Extractions for
protein or total RNA isolation were either done immediately after culture or after

quick freezing in liquid Nitrogen and storage at -80° C.

Cloning of Actin cDNA

A partial genomic clone encoding the Drosophila actin 50 gene was
kindly provided by Dr. Eric Fryberg (Fryberg et al., 1980). This DNA fragment
was labeled with [32F] using the random primer method and used to screen a A.
gt11 Aedes aegypti cDNA library made from vitellogenic fat body mRNA (Cho et
al., 1991) as outlined by Sambrook et al. (1989). Several positive clones were
obtained and subcloned into pUC119 for sequencing. Northern blots of
mosquito total RNA probed with the putative mosquito actin cDNA or the
Drosophila actin gene showed hybridization to the same band. In addition, the
deduced amino acid sequence showed > 85% identity with the Drosophila actin
gene, confirming the identity of the mosquito actin cDNA.

85
In vitro Fat Body Culture

Abdomens from blood-fed or three day old sugar-fed females were
isolated and placed in a tissue culture system as previously described (Koller et
al., 1989; Dhadialla and Raikhel, 1990). Protein was labeled by including [358]
methionine in the culture media. 20E (Calbiochem) was dissolved in ethanol
and stored at -80° C until immediately before use. Chx (Calbiochem) was
dissolved in H20 immediately before use. Radiochemicals were purchased from
Amersham and New England Nuclear. All tissue culturing was performed in a
hood using sterile instruments to reduce contamination. Cultures were
incubated at 27° C for the duration of the experiments. Three repetitions of 9 fat

bodies were cultured for each treatment.

Protein Analysis

Cellular proteins were isolated by homogenizing cultured fat bodies in B1
buffer (50mM Tris pH 8.4, 50mM NaCl, 0.2% Triton-X-100, 0.02% sodium azide).
Vg was immunoprecipitated using monoclonal antibodies specific to both the
large and small Vg subunits. Vg protein levels were estimated by measuring the
level of [35S] in the precipitate using a scintillation counter (LKB Wallace)
(Raikhel and Bose, 1988; Dhadialla and Raikhel, 1990). Total protein levels
were calculated by trichloroacetic acid precipitation and subsequent scintillation
counting of incorporated [35S]. All experiments were done in triplicate to

ensure reproducibility.

mRNA Analysis
Cultured fat bodies were homogenized and total RNA isolated as
previously described (Raikhel and Bose, 1988; Cho et al., 1991). RNA

concentration was measured using a spectrophotometer. Equal amounts of

86

RNA were blotted onto 0.45pm nitrocellulose membrane (MSI) using a Bio-Rad
slot blot apparatus and the protocol described by Sambrook et al. (1989). All
RNA samples were divided into three equal portions and blotted separately.
These replicate blots were probed with random primer labeled cDNA clones for
Vg, VCP or actin as described (Cho et al., 1991) and used to expose film at -70°
C using an intensifying screen. After autoradiography, the blots were cut into
pieces and each individual sample placed in a scintillation counter for
quantification. All experiments were done in triplicate to ensure reproducibility.
To ensure that the above procedure accurately quantifies different levels
of probe hybridization, the following pilot experiment was done. Serial dilutions
of the cDNAs for VCP, V9 and actin were slot blotted, baked, then hybridized to
radiolabeled cDNA probes. The blots were autoradiographed, cut into pieces
and each sample placed in a scintillation counter for quantification. The results
showed that this method accurately determined the level of cDNA blotted onto

the membrane to within + or - 7 %.

87
RESULTS

Initiation of Transcription and Syn thesis of Vitellogenin and VCP by
Cultured Fat Bodies in Response to 20E

The effect of 20E on the initiation of yolk protein production in the fat body
of female mosquitoes was first investigated. The level of Vg production was
measured in cultured previtellogenic fat bodies in response to increasing
amounts of 20E added to the culture media. Fat bodies from sugar-fed females
were incubated for 6 hrs in culture media containing [358] methionine and
different concentrations of 20E with a change of media every 2 hrs. Fat bodies
from sugar-fed females cultured in media containing no 20E were used to
establish a background level of expression while fat bodies from blood-fed
females 6 hrs after feeding were used to show a 100% response. V9 and total
protein production reached maximum levels at 10'5 M 20E (Fig. 1). V9 levels
reached ~60% of that obtained from blood-fed controls while total protein levels
reached ~87% of control. Concentrations greater than 10'5 M did not give any
further increases in V9 or total protein production (not shown).

The effect of 20E on the initiation of yolk protein gene transcription in
cultured previtellogenic fat bodies was then examined. This was done by
measuring the amount of V9 and VCP mRNA present in fat bodies from sugar-
fed females incubated for 6 hrs in culture media (changed every 2 hrs)
containing different concentrations of 20E. V9 and VCP mRNA produced by fat
bodies from blood-fed females 6 hrs post blood meal was measured to
determine in vivo levels of mRNA. The amount of actin mRNA produced also
was measured to show a basal level of transcription of a non-vitellogenic gene.
Both V9 and VCP mRNA are transcribed in response to 20E, reaching maximum

transcription rates at 10"5 M (Fig. 2), concentrations similar to those inducing

Fig. 1. Effects of 20E concentrations on V9 and total protein production in
cultured fat bodies. Previtellogenic fat bodies were incubated for 6 hrs in
culture media containing 0 to 10'5M 20E and [35S] methionine as described in
Materials and Methods. Fat bodies taken from blood-fed females were used as
a positive control. After culturing, proteins were extracted from the tissue and V9
immunoprecipitated using monoclonal antibodies specific to this protein. Total
protein was precipitated with TCA. The activity was determined using a
scintillation counter. The vertical line at the top of each bar indicates the

standard error of three repetitions.

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Fig. 2. Effect of 20E concentration on Vg, VCP and actin mRNA production
in cultured fat bodies. Cultured previtellogenic fat bodies were incubated for 6
hrs with 0 to 10'4 M 20E. Total RNA was extracted and levels of Vg, VCP and
actin mRNA measured by slot blot and hybridization to the appropriate
radiolabeled cDNA probes. Fat bodies from females 6 hrs after a blood meal
were used to show an in vivo response. Hybridization was quantified using a
scintillation counter. Vertical lines indicate the standard error of the mean of

three repetitions.

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maximum production of V9 protein. At this concentration, V9 and VCP mRNA
levels reached ~81% and 82% of that obtained from blood-fed controls,
respectively. Background levels of Vg mRNA were always found to be higher
than VCP levels in previtellogenic fat bodies and controls untreated with 20E,
indicating that a low level of V9 transcription is present in non-induced fat

bodies. As expected, actin levels remained relatively constant.

Effect of Cycloheximide on Cultured Fat Bodies

The minimum concentration of Chx capable of inhibiting the majority of
protein synthesis was determined using a dose response curve of protein
production in response to increasing concentrations of Chx. Fat bodies from
females 18-20 hrs after a blood meal (near the peak of yolk protein production)
were treated with different doses of Chx for 1 hr, then incubated in media
containing Chx and [35S] methionine for 1 hr to label newly synthesized
proteins. At 10'4 M Chx, >98% of protein synthesis is inhibited (Fig. 3),
providing a treatment concentration for subsequent experiments.

The effect of Chx on yolk protein mRNA transcription was now tested by
culturing sugar-fed female fat bodies in media containing 10‘4M Chx for 1 hr,
then in media containing 10'5M 20E and 10'4M Chx for 6 hrs. The media was
changed every 2 hrs. 10'4 M Chx virtually eliminated induction V9 and VCP
gene transcription in response to 20E (Fig. 4). At the same time, Chx appears to
have little effect on the transcription rate of the actin gene. Background Vg
transcription was again higher than that of VCP. To ensure that this inhibition
was not due to a decay of cellular proteins needed for the 20E response over
the 6 hr incubation, RNA was extracted from fat bodies cultured as above after
only 2 hrs of 20E stimulation. This experiment also showed elimination of 20E

induction of VCP and V9 transcription (not shown). Thus, these experiments

93

Fig. 3. Inhibition of V9 and total protein synthesis by cycloheximide.
Vitellogenic fat bodies 18 hrs post blood meal were cultured for 1 hr in 0 to 10'
3M Chx followed by 1 hr with Chx and [35S] methionine. Levels of newly
synthesized V9 and total protein were measured as described in the text.
Values are expressed as percentages of that produced by fat bodies cultured
without Chx (0 M Chx, control).

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Fig. 4. Effect of Chx on Vg, VCP and actin mRNA production.
Previtellogenic fat bodies were preincubated with or without Chx for 1hr, then
cultured for 6 hrs in media containing different combination of Chx and 20E. Fat
bodies taken from previtellogenic sugar-fed females were used as a control.
After culturing, total RNA was extracted and levels of Vg, VCP and actin mRNA
measured by slot blot and hybridization to the appropriate radiolabeled cDNA
probes. A. Quantitation of hybridization using a scintillation counter. Vertical
lines show the standard error of the mean of three repetitions. B.

Autoradiograph of slot blots probed with cDNAs for Vg, VCP and actin.

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indicate that inhibition of protein synthesis abolishes the ability of 20E to induce

yolk protein transcription.

Recovery of Yolk Protein Production and Transcription After
Cycloheximide Treatment

To ensure that the effect of Chx on protein synthesis is not simply the
result of cell death, the following recovery experiments were done. First, fat
bodies from females 18 hrs after a blood meal were incubated in media
containing Chx for 1 hr, rinsed twice with culture media, then incubated for 1 hr
in standard culture media, followed by the labeling of newly synthesized proteins
for 1 hr in culture media containing [358] methionine. Both total protein
synthesis and V9 synthesis were recovered by washing the Chx from the tissue
(Fig. 5). At concentrations of both 10'4 and 10'5M Chx, >90% of protein
synthesis is inhibited, yet significant synthetic capability can be recovered within
2 hr of removing the Chx from the culture media, indicating that these
concentrations of Chx induce a reversible inhibition of protein synthesis. The
cultures treated with 10'5 M Chx recovered more rapidly or more completely
(~60% of control) than those treated with 10'4 M (~40% of control). This is likely
because Chx is more readily removed from cultures where the initial
concentration is lower.

Initiation of transcription of V9 and VCP mRNA in previtellogenic fat
bodies can also be recovered by removal of Chx from the culture media.
Previtellogenic fat bodies were incubated in culture media with 10‘4M Chx for 1
hr, rinsed twice in standard culture media then incubated in culture media with
10’5M 20E for 6 hrs, changing the media every 2 hrs. The cultures rinsed of
Chx showed significant levels of V9 and VCP mRNA, indicating the tissue can

99

Fig. 5. Recovery of vitellogenesis after removal of Chx. V9 and total protein
produced by cultured vitellogenic fat bodies 18 hrs post blood meal under
different conditions: 1, culture media plus 104M Chx for 3 hrs; 2, culture media
plus 10'4 M Chx for 1 hr, 0M Chx for 2 hrs; 3, culture media plus 10'5M Chx for
3 hrs; 4, culture media plus 10'5M Chx 1 hr, 0M Chx for 2 hrs; 5, culture media
with no Chx for 3 hrs. In all treatments, protein production was measured for the
third hour of incubation. Values are expressed as a percentage of levels
produced by tissue cultured without Chx (treatment 5, control). Vertical lines

indicate the standard error of the mean of three repetitions.

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initiate transcription of these genes in response to 20E after Chx has been
removed (not shown). Actin mRNA levels again appear relatively constant.

Last, recovery of the ability of cultured previtellogenic fat bodies to
respond to 20E after 6 hrs of Chx treatment was considered. Fat bodies from
previtellogenic females were incubated in media containing Chx for 6 hrs, rinsed
three times with culture media containing 10‘5M 20E over a 1 hr period, then
incubated for 1 hr in culture media containing [358] methionine/10'5M 20E,
followed by a 1 hr incubation in standard culture media/10'5M 20E to obtain
labeled secreted proteins. The culture media was TCA precipitated to measure
protein production and the tissue homogenized for RNA analysis. Both protein
synthesis and yolk protein mRNA transcription were recovered by washing the
Chx from the tissue. Within 3 hrs of removal of the Chx from the media, protein
production had returned to 70% of the untreated control. Induction of both V9
and VCP mRNA was detected in cultures rinsed of Chx (Fig. 6), indicating the
cultured tissue is still capable of responding to 20E even after the 6 hr Chx

treatment. Actin levels again appear relatively constant.

102

Fig. 6. Recovery of V9 and VCP mRNA production after removal of Chx.
Autoradiograph of slot blots probed with cDNAs for Vg, VCP and actin.
Previtellogenic fat bodies were incubated in culture media containing 104M Chx
for 1 hr, rinsed of Chx, then incubated in culture media free of Chx and
containing 10'5M 20E for 6 hrs. Total RNA was extracted and levels of Vg, VCP
and actin mRNA measured by slot blot and hybridization to the appropriate
cDNA probes. Top lane: RNA from cultured fat bodies treated with 10‘5M ZOE
only; center lane: RNA from cultured fat bodies treated with 10'4M Chx and then
in 10'5M 20E in the presence of Chx; last lane: RNA from cultured fat bodies
treated with 10'4 M Chx, rinsed in culture media without Chx and then treated
with 10‘5M 205 only.

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1 04
DISCUSSION

The effect of hormones on gene expression is of fundamental interest to
the field of developmental biology. A great deal of research has focused on the
mode of action of numerous hormones as they illicit transcription of their target
genes or as they initiate a hormone response cascade. Within such a cascade,
the hormone acts on particular genes both directly and indirectly. Direct effects
on gene expression can be defined as those which are rapid and can occur even
though synthesis of new proteins has been inhibited (Yamamoto and Alberts,
1976). An example of this is the glucocorticoid induction of MMTV RNA (Ringold
et al., 1977). Conversely, an indirect response is one in which the initial effect of
the hormone is thought to be mediated or amplified by production of other
regulatory proteins. Such control would consequently be sensitive to inhibition
of protein synthesis. Examples of indirect responses include the induction of
the late gene products in Drosophila salivary glands by 20E (Ashbumer, 1973)
and the estrogen induction of ovalbumin mRNA in the chick oviduct (McKnight
and Palmiter, 1979).

In Xenopus Ieavis, the liver produces high levels of V9 in response to
estrogen. Experiments using Chx showed that the effect of estrogen on V9
mRNA production is direct because inhibition of liver protein synthesis does not
prevent activation of V9 gene transcription (Hayward et al., 1982). The action of
20E on the yolk protein genes in files appears to be quite different. In
Drosophila, Bownes et al. (1987) used Chx to determine the nature of the effect
of 20E on yolk protein transcription. Their results indicated that protein
synthesis is required for 20E to initiate and maintain yolk protein transcription.
However, these studies were done in vivo and secondary effects of Chx on the

insect cannot be ruled out. By using a well defined in vitro system, the relevant

105

tissue can be isolated and a more controlled experiment done. Our studies
indicate that in the mosquito 20E is acting on the genes for V9 and VCP
indirectly. 20E induces transcription of these genes in a dose dependent
manner (Fig. 2), yet protein synthesis inhibition completely eliminates this
induction (Fig. 4). It is unlikely that the effect of Chx on V9 and VCP
transcription is due to decay of the 20E receptor over the course of the
experiment, rather than the interuption of the 20E response pathway because
Chx treatment leads to an immediate elimination of V9 and VCP transcription in
response to 20E rather than a slow decline, and this response is rapidly
recovered upon removal of Chx. In addtion, similar experiments with cultured
Drosophila salivary glands showed that proteins required for direct responses to
20E are present through 8 hrs of Chx treatment, indicating that the 20E receptor
persists throughout such an experiment (Segraves and Hogness, 1990). Thus, a
20E response cascade is likely being induced by rising titers of the hormone with
the genes for V9 and VCP downstream in the hierarchy.

This conclusion is consistent with the recent finding that upstream of the
VCP gene exists a putative response element that shows limited similarity to the
consensus ecdysone receptor binding site (Deitsch and Raikhel, 1993). This
palindromic sequence is found tandemly repeated four times and has no spacing
between half sites. This is in contrast to the one base pair spacing shown to be
necessary for binding of the ecdysone receptor in Drosophila (Antoniewski et al.,
1993). The cDNA for the ecdysone receptor from mosquito has recently been
cloned and shows very high similarity to that of Drosophila, particularly in the P-
box domain (100% amino acid identity), indicating that its binding specificity will
also likely depend on a one base pair spacer (Kapitskaya et al., 1993).

Therefore, it is likely that the putative response element upstream of VCP is

106

bound by a transcription factor other than 20E. Elucidation of this activating
protein awaits further research.

The conclusion that the control of the yolk protein genes by ZOE in
mosquito is indirect provides the necessary groundwork for future research in
this area. By eventually uncovering the entire hierarchy of target genes and
activator proteins, a more complete understanding of the effect of 20E on the

development of mosquitoes and insects in general can be acquired.

107

ACKNOWLEDGEMENTS

We thank Dr. Tom Sappington and Dr Suzanne Thiem for their critical
reading of the manuscript. This research was supported by grant Al-24716 from
the National Institutes of Health to A.S.R.

108
REFERENCES

Antoniewski 0., Laval M. and Lepesant J. (1993) Structural features critical to
the activity of an ecdysone receptor. Insect Biochem. Mol. Biol. 23, 105-
1 14.

Ashbumer M. (1973) Sequential gene activation by ecdysone in polytene
chromosomes of Drosophila melanogaster. Dev. Biol. 35, 47-61.

Ashbumer M., Chihara C., Meltzer P. and Richards G. (1974) Temporal control
of puffing activity in polytene chromosomes. Cold Spring Harbor Symp.
Quant. Biol. 38, 655-662.

Bownes M., Scott A. and Blair M. (1987) The use of an inhibitor of protein
synthesis to investigate the roles of ecdysteroids and sex-determination
genes on the expression of the genes encoding the Drosophila yolk
proteins. Development 101,931 -941 .

Burtis K, Thummel C., Jones C., Karmin F. and Hogness D. (1990) The
DroSOphiIa E74F early puff contains E74, a complex ecdysone-inducible
gene that encodes two ets-related proteins. Cell 61, 85-99.

Chen J-S., Cho W-L. and Raikhel A. (1994) Analysis of the cDNA for mosquito
vitellogenin: similarity with vertebrate phosvitins and arthropod serum
proteins. J. Mol. Biol. 237, 641 -647.

Cho W., Deitsch K and Raikhel A (1991) An extraovarian protein accumulated
in mosquito oocytes is a carboxypeptidase activated in embryos. Proc.
Natl. Acad. Sci. USA 88, 10821-10824.

Deitsch K and Raikhel A. (1993) Cloning and analysis of the locus for mosquito
vitellogenic carboxypeptidase. Insect Mol. Biol. 2(4) In press.

Dhadialla T. and Raikhel A. (1990) Biosynthesis of mosquito vitellogenin. J.
Biol. Chem. 265, 9924-9933.

Fryberg E., Kindle K, Davidson N. and Sodja A. (1980) The actin genes of
DrOSOphila: a dispersed multigene family. Cell 19, 366-378.

Fuchs M. and Kang S-H. (1981) Ecdysone and mosquito vitellogenesis: a
critical appraisal. Insect Biochem. 11, 627-633.

109

Hagedom H. (1983) The role of ecdysteroids in the adult insect. In
Endocrinology of Insects (edited by Downer R. and Laufer H.), pp. 271-
304. Alan R. Liss, Inc., New York.

Hagedom H. (1985) The role of ecdysteroids in reproduction. In Comprehensive
Insect Physiology, Biochemistry and Pharmacology (edited by Kerkut G.
and Gilbert K), vol. 8, pp. 205-261. Pergamon Press, Oxford.

Hagedom H., Turner 8., Hagedom E., Pontecorvo D., Greenbaum P., Pfeiffer D.,
Wheelock G. and Flanagan T. (1977) Post-emergence growth of the
ovarian follicles of Aedes aegypti. J. Insect Phys. 23, 203-206.

Hays A and Raikhel A (1990) A novel protein produced by the vitellogenic fat
body and accumulated in mosquito oocytes. Roux's Arch. Dev. Biol. 199,
1 14-121.

Hayward M., Brock M. and Shapiro D. (1982) Activation of vitellogenin gene
transcription is a direct response to estrogen in Xenopus Iaevis liver.
Nucleic Acids Res. 10, 8273-8284.

Kapitskaya M., Cho W-L. and Raikhel A (1993) Analysis of mosquito
ecdysteroid receptor cDNA and expression during vitellogenesis. Second
Int. Symp. on Mol. Insect Sci. pg. 87.

Koelle M., Talbot W., Segraves W., Bender M., Cherbas P. and Hogness D.
(1991) The Drosophila EcR gene encodes an ecdysone receptor, a new
member of the steroid receptor superfamily. Cell 67, 59-77.

Koller C., Dhadialla T. and Raikhel A (1989) Selective endocytosis of
vitellogenin by oocytes of the mosquito, Aedes aegypti: an in vitro study.
Insect Biochem. 19, 693-702.

Lea A. (1964) Studies on the dietary and endocrine regulation of autogenous
Aedes taeniorhynchus. J. Med. Ent. 1 , 40-44.

Lea A. (1982) Artifactual stimulation of vitellogenesis in Aedes aegypti by 20-
hydroxyecdysone. J. Insect Physiol. 28, 173-176.

McKnight G. and Palmiter R. (1979) Transcriptional regulation of the ovalbumin
and conalbumin genes by steroid hormones in chicken oviduct. J. Biol.
Chem. 254, 9050-9058.

Racioppi J., Gemmill R., Kogan P., Calvo J. and Hagedom H. (1986)
Expression and regulation of vitellogenic messenger RNA in the mosquito
Aedes aegypti. Insect Biochem. 16, 255-262.

 

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Raikhel A (1992) Vitellogenesis in mosquitoes. In Advances In Disease Vector
Research. vol. 9, pp. 1-39. Springer-Verlag, New York.

Raikhel A. and Bose S. (1988) Properties of the mosquito yolk protein: a study
using monoclonal antibodies. Insect Biochem. 18, 565-576.

Raikhel A and Lea A. (1983) Previtellogenic development and vitellogenin
synthesis in the fat body of a mosquito: an ultrastructural and
immunocytochemical study. Tissue Cell 15, 281-300.

Ringold G., Yamamoto K, Tomkin 6., Bishop J. and Vannus H. (1977)
GIucocorticoid-stimulated accumulation of mouse mammary tumor virus
RNA: increased rate of synthesis of viral RNA. Proc. Natl. Acad. Sci. USA
74, 2879-2883.

Sambrook J., Fritsch E. and Maniatis T. (1989) Molecular Cloning: A Laboratory
Manual, 2’79 Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor,
NY.

Segraves W. and Hogness D. (1990) The E75 edcysone-inducible gene
responsible for the 75B early puff in Drosophila encodes two new
members of the steroid receptor superfamily. Genes Dev. 4, 204-219.

Yamamoto K and Alberts B. (1976) Steroid receptors: elements for modulation
of eukaryotic transcription. Ann. Rev. Biochem. 45, 721-746.

111

CHAPTER 5

SUMMARY AND FUTURE RESEARCH PROSPECTS

 

112
SUMMARY

1 . VCP cDNA cloning and analysis:

Using antibodies to a purified 53 kDa yolk protein from the female
mosquito, Aedes aegypti, a partial cDNA was cloned from a kgt11 cDNA library
made from mRNA isolated from vitellogenic fat bodies. This initial 840 bp clone
was used to rescreen the library and a 1020 bp clone containing the 5' end of
the cDNA was isolated. The remaining 3' end of the cDNA was obtained using
PCR. The complete nucleotide sequence of the cDNA was determined using the
dideoxy sequencing method. Northern analysis was done using the isolated
cDNA fragment as a probe to demonstrate the stage-, sex- and tissue- specific
expression expected for a gene encoding a mosquito yolk protein. Final
confirmation of the identity of the clone was obtained by comparison of the
deduced amino acid sequence from the cDNA to that provided by direct N-
tenninal sequencing of the purified protein. Comparison of the complete
deduced amino acid sequence to those found in the data base indicated strong
homology to members of a carboxypeptidase family. This strongly indicated that
this yolk protein was an enzyme made by the maternal fat body and stored in the
developing oocyte for use during embryonic development. Experiments using
the serine protease inhibitor DFP confirmed that this protein is in fact a
proenzyme produced by the vitellogenic fat body, stored in the egg, then
subsequently activated in the developing embryo, showing its highest level of
activity during mid-embryogenesis. This protein was therefore named

Vitellogenic Carboxypeptidase (VCP).

 

113

2. VCP gene cloning and analysis:

In order to investigate the regulation of expression of the gene for VCP,
the cDNA was used as a probe to screen a A phage library made from genomic
DNA extracted from the colony of mosquitoes maintained in our laboratory.
Numerous clones were obtained and restriction mapped. The maps indicated
that the cloned came from two distinct groups, representing either two separate
alleles of the VCP gene within the population or two copies of the gene within
the genome. Genomic blots showed fragments representing both VCP clones in
equal proportions. However, blots made from DNA taken from individual
mosquitoes showed that the two different clones clearly represent alleles, as
homozygotes for each allele exist as well as the corresponding heterozygotes.
Both alleles of the gene were sequenced in both directions using the dideoxy
method and the nucleotide sequence analyzed both visually and with the aid of a
computer. Several potential control elements were found surrounding the coding
region. A putative TATA box and arthropod initiator were found near a proposed
transcription start site. Primer extension analysis confirmed that transcription
initiation does indeed begin in this region. Upstream of this site exists a 16 bp
imperfect palindrome repeated 4 times. This sequence shows similarity to
previously defined steroid hormone response elements. In addition, several
other sequences were identified that are similar to regulatory elements found in
other organisms, including a downstream element with similarity to a Drosophila
fat body enhancer. The function of these putative regulatory elements awaits

further analysis. ~

3. Control of yolk protein expression by 20E:
The vitellogenic cycle of female mosquitoes has been shown to be

initiated with a rise in the titer of the steroid hormone 20-hydroxyecdysone (20E).

 

114

How this hormone affects the rate of transcription of the yolk protein genes has
been a matter of debate. Using the protein synthesis inhibitor cycloheximide
(Chx), and an in vitro culture system of mosquito fat bodies, we attempted to
determine whether the effect of 20E on V9 and VCP transcription was direct or
indirect. First, the optimum titer of 20E needed in culture to illicit maximum
levels of yolk protein production was determined. Then the concentration of
Chx needed to reversibly inhibit > 98% of protein production by the cultured fat
body was established. With these experimental conditions known, transcription
of the genes for V9 and VCP in the presence of 20E with or without Chx in the
media was measured. The results indicated that protein synthesis is required in
response to 205 before transcription of the yolk protein genes is initiated. This
is indicative of a hormone response cascade existing with one or more steps
between the 20E/receptor complex and the genes for V9 and VCP. Such a
cascade may resemble that proposed by Ashbumer when considering the

activation of 20E responsive genes in Drosophila salivary glands.

1 15
FUTURE RESEARCH PROSPECTS

1 . DNA structure sunounding the gene for VCP:

It was discovered in the late 1960's that eukaryotic genomes contain
highly repeated, moderately repeated and unique genomic sequences (reviewed
in Britton and Kohne, 1968). The structure of the repetitive DNA can be
arranged in three different classes; 1) simple repetitive DNA, usually shorter
than 600 bp, interspersed with single- or few-copy segments, 2) DNA organized
as long tandem arrays of identical repeating units, and 3) stretches of repetitive
DNA in which short sequences are interspersed with one another in complex
permutations (Kumar and Rai, 1991). These repeated sequences seem to be
maintained through evolution and many such DNA fragments have been found
to hybridize to genomic DNA isolated from a number of diverse species. This
has been suggested to infer a sequence-dependent function for such families of
repetitive DNA (Besansky, 1992). It has also been suggested that the
sequences are parasitic elements capable of independently replicating in the
genome and survive the pressure of natural selection without fulfilling any useful
function (Orgel and Crick, 1980). Some repetitive elements interspersed in the
gamma are mobile and their ability to transpose can lead to gross evolutionary
changes through genomic rearrangements (Georgiev et al., 1982).

Mosquitoes show a wide range of repetitive DNA content within their
genomes, ranging from 20% in Anopheles quadrimaculatus to 84% in Aedes
tn'sen'atus( Black and Rai, 1988). It was found that the proportion of the genome
occupied by repeated DNA increases as genome size increases. It was also
found that the amplification of short, interspersed repeats accounts for, at least
in part, the increased genome size. This is in contrast to Drosophila

melanogaster in which repetitive DNA is often clustered (Manning et al., 1975).

116

The species of mosquito used in our laboratory, Aedes aegypti, has a relatively
large genome, and as a result, would be expected to show a fairly high level of
interspersed repetitive DNA.

When the 2 alleles of VCP were cloned and analyzed, a region of very
low homology was found downstream of the coding region in both VCP1 and
VCP2. Partial sequence analysis showed virtually no homology between these
two elements, and cross hybridization of the two clones to one another showed
that these unique regions begin 110 bp downstream of the polyadenylation
signal and extend ~3 kb, after which homology resumes. At first, these non-
homologous elements were thought to be artifacts of cloning (i.e. recombinant
clones that occurred when the library was made) that did not reflect the actual
structure of the mosquito genome. However, two lines of evidence argue
against this: 1) numerous, overlapping clones of various lengths were obtained
for both VCP1 and VCP2. All clones contain these unique elements. It is
unlikely that such a recombination event would have occurred in each clone
independently, and 2) Southern blots of genomic DNA digested with enzymes
that cut within the coding region and within the non-homologous region
produced bands of the size predicted by the restriction maps of the cloned DNA.
This indicates that the clones have the same restriction map as the isolated
genomic DNA Thus it appears that the clones VCP‘I and VCP2 do in fact reflect
the structure of the locus for VCP.

Next, the structure of these unique elements themselves were considered.
When a small fragment of the 5' end of the element found in VCP2 was labeled
and hybridized with Southern blots of both VCP1 and VCP2, it hybridized only
weakly with a fragment from VCP1, but hybridized with 2 fragments in VCP2
(Fig. 1). This indicates that this DNA fragment is repeated within the unique

region downstream of the VCP1 coding region. Similar results were obtained

117

Fig. 1: Southern blots showing the repetitive nature of the DNA In the
region downstream of the VCP locus. A. [5le digests of VCP'I (left) and
VCP2 1 clones run on a 1% agarose gel and stained with EtBr. B. Southern blot
probed with the 5' end of the unhomologous region from VCP2. C. Genomic
DNA cut with EcoRI, run on a 0.8% gel and probed with the 5' end of the
nonhomologous region from VCP2. D. Restriction map of VCP2 showing the

probe and the hybridizing fragments.

118

 

~2.l kb insert

I? = EcoRl t‘probe hybridizing fragments

119

when the same experiment was done using a small fragment of the unique
region downstream of VCP1. These experiments suggest that these three kb
unique regions are either simple sequences of tandemly repeated DNA, or
perhaps that they have repeated DNA at both ends of the element, much like the
LTR's of many transposable elements. When fragments from the regions were
labeled and used to probe genomic blots, high molecular weight smears
resulted, indicating that the sequence is highly repeated within and dispersed
throughout the genome (Fig. 1) (Kumar and Rai, 1991).

One of these elements is currently being sequenced to determine its
exact structure. Such repetitive elements are often used in the determination of
species and in studies of genome evolution (Besansky, 1992). These unique

DNA elements may prove to be very interesting fragments of DNA.

2. Development of an expression system:

One of the most powerful methods of analyzing the regulatory regions of a
particular gene is through the use of an expression system. In this method, a
piece of DNA containing the regulatory region of the gene of interest is
introduced into the system and the level of transcription measured. Once
transcription is established, either the reaction conditions or the DNA itself can
be altered to determine the important components involved in transcriptional
regulation of the gene being investigated. Many systems have been developed
for this purpose, including in vitro transcription (Morton and Sprague, 1984),
stable and transient cell culture transformation (Fallon, 1986) and germline
transformation (Spradling and Rubin, 1982). In insects, by far the most
advanced system developed thus far is in Drosophila, where the transpcsable
element P is used to introduce foreign DNA directly into the germline. This

results in a stabley transformed line of transgenic flies, with the introduced DNA

120

being inherited in a Medellian fashion. This system has been used to
investigate the regulation of numerous genes in fruit flies.

Outside of Drosophila, no other system of routine germline transformation
has been developed in insects. Alternatively, some expression of exogenous
DNA has been observed in cell culture (Fallon, 1986). This system is somewhat
limited, however, because the cell lines thus far available are embryonic in origin
and as a result, undifferentiated. Thus, any sex-, tissue-, or sex-specific genes
are unlikely to be correctly expressed if introduced into such cells. In fact, a
construct containing the regulatory region of the mosquito Vg gene introduced
into a mosquito cell line showed very low expression, even in response to
treatment with 20E (Fallon and Hagedom, unpublished). Until a cell line
expressing female vitellogenic genes has been established, it is unlikely that use
of a cell culture system will be an attractive method for analysis of the VCP
regulatory region. As an alternative, organ culture systems have been
developed as a way to separate differentiated tissue from the organism for more
detailed biochemical and physiological experiments. It is possible that
transformation of such cultured cells will provide a method for transient
expression of exogenous DNA.

Liposomes have been used as a way to introduce foreign DNA into cells
(Fraley et al., 1981). In this method, a synthetic cationic lipid is used to make
small, unilamellar liposomes that spontaneously interact with DNA to form lipid-
DNA complexes. These complexes subsequently fuse with the plasma
membrane of the cells, introducing the encapsulated DNA While providing no
basis for DNA integration or transcriptional regulation, this provides an efficient
and convenient method for getting a DNA construct into target cells. As a result,
it is now widely used to introduce DNA into cultured cells for transient expression

assays where stable transformation is either not yet possible or not convenient.

121

Recently, liposome transfection has been used to introduce foreign DNA into
mouse cells in vivo (Brigham, et al., 1989). In this particular experiment, the
liposome-DNA complex was injected into the blood stream of the animal, and
subsequently showed expression in the lungs. This seems to indicate that this
method may be applicable to intact animals or tissue culture systems.

In mosquitoes, it was recently reported that liposome-transfection was
used to introduce a construct into cultured salivary glands (Morris and James,
1993). A Drosophila heat-shock promoter, previously shown to be active in
mosquito cells, was used to drive a Iuciferase reporter gene. The cultured
glands were first treated with elastase to remove the basement membrane, then
transfected using the liposome-mediated technique. Luciferase activity was
detected, indicating the foreign DNA was taken-up by the cells and expressed.
This technique may be applicable to VCP analysis. A fat body tissue culture
system has been well established and the conditions needed to induce
expression of VCP have been defined (Chapter 4). The VCP regulatory region
should be an excellent candidate for developing such an expression system do
to its tight regulation and its high level of expression when induced. For this
purpose, the construct shown in Fig. 2 has been made and will be used in
experiments aimed at developing a transcriptional assay. The construct
contains 1700 bp of DNA upstream of the start site, including the arthropod
initiator, TATA box, putative ribosomal binding site and the 4 repeated imperfect
palindromes resembling hormone response elements. Downstream of the
reporter are 800 bp of the VCP downstream region, including a small stretch of
sequence resembling the Drosophila fat body enhancer (Garabedien, et. al.,
1986). If transcription can be established using this system, deletions and/or
site directed mutagenesis will be used to define those sequences surrounding

the VCP gene that are responsible for control of its expression.

122

Fig. 2: Diagram of the construct to be used In lipofection experlments.
This construct was made by inserting the 1700 bp 5' upstream region from VCP1
into the (name of vector) polycloning site. Downstream of the Iuciferase reporter
gene was cloned the 3' region of VCP1 containing a region of similarity to the
Drosophila fat body enhancer. Also including in the vector are and SV40 poly(A)
signal, an ampicillan resistance gene for propagation in E. coli, and a poly(A)
signal downstream of the ampr gene to prevent raadthrough transcription from

the ampr promoter.

123

Ampr \‘ I/ POI?“

        
  
 
 
 

-----
..........................

. a'o'l‘ ............................... .

.......................

........................

 

.v.
.....

1700 bp VCP upstream

r ion
500 bp VCP 99

downstream regio\n'
3V4! 51‘

POIYW

luciferase
reporter gene

1 24
3. Analysis of DNA/protein interactions:

Once a region of DNA has been shown to be active in inducing
transcription of a particular gene, often it is interesting to know exactly what base
pairs are bound by a particular protein, which are important for DNA binding,
how tightly the protein binds to the DNA, and what the identity of the regulatory
protein is. Often these questions are addressed using footprinting (Carthaw et
al., 1986) or gel-retardation (Fried and Crothers, 1981) assays. In gel-
retardation assays, a labeled fragment of DNA that is thought to be bound by a
regulatory protein is incubated in either a crude nuclear extract or in the
presence of the purified DNA binding protein. After the protein is allowed to bind
to the DNA, the complex is loaded onto an acrylamide gel and electrophoresad
to separate bound and unbound DNA. DNA bound by the regulatory protein has
its migration through the gel "retarded". Competition assays using cold
competitor DNA can be used to show specificity of the interaction and to
deterrnina binding affinity of the protein. Usually, a synthetic nucleic acid such
as polydl-polydC is used to eliminate non-specific binding. In this way, gel-
retardation assays can be a useful technique in identifying important regulatory
regions controlling transcription of a particular gene.

Footprinting involves much the same idea as above, except that rather
than electrophoresing a DNA/protein complex, this interaction is detected by
digesting the DNA with either a nuclease, such as DNAsel (Carthaw at al.,
1985), or with a chemical such as hydroxyl radicals (Tullius, 1990). The DNA
bound by the protein is protected from this digestion, and the protected region is
detected on a subsequent sequencing gel. The DNA is end-labeled before the
reaction, thus when cleaved, it will produce a ladder of bands on a sequencing
gel. Spaces, or “footprints", in this ladder are left where the DNA has been
protected by bound protein. In this way, this technique can detect the specific

125

base pairs bound by the regulatory protein. Altering the binding conditions or
mutating the sequence of the DNA can provide information about what base
pairs or conditions are necessary for binding.

Once a specific DNA sequence has been identified as being bound by a
specific regulatory protein, it is possible to use this sequence to either purify the
protein (Rutberg and Ronai, 1992) or to directly clone the cDNA encoding this
protein from an expression library (Vinson, at al., 1988; Shea, at al., 1990).
Affinity columns can be made using the binding sequence from the regulatory
region of a gene as the ligand. Nuclear extracts from tissue actively transcribing
the gene are then passed over the column under conditions previously shown to
promote DNA/protein interaction. The column is then washed and the highly
enriched protein eluted. The characteristics of the purified protein can then be
studied or it can be used in the first steps of cloning. Altamatively, a labeled
fragment of DNA identified as a binding site can be used as a probe to screen
nitrocellulose lifts of proteins produced by clones in an expression library. The
DNA should be bound by the regulatory protein of interest and, in this way, the
cDNA for this protein is obtained. This is limited to proteins that bind as
monomers or homopolymers, and also tends to identify numerous non-specific
DNA binding proteins. It has, however, been used to clone several regulatory
proteins.

Some of these techniques will undoubtedly be used in the future analysis
of the regulation of the VCP gene. Perhaps the most immediately available
procedure would be to use several fat body specific regulatory proteins recently
cloned from Drosophila, including a member of the CREB/ATF family (Abel, et
al., 1992) and AEF-I (Falb and Maniatis, 1992), and to attempt gel-retardation
assays using them. The conditions for binding of these proteins, produced in E.

coli, have been detarrninad. Also, the mosquito homologue of a liver-specific

126

regulatory protein (HNF-4) has also been recently cloned (Kapitskaya and
Raikhel, unpublished). Binding of this protein to the VCP regulatory region
could also be tried. Altamatively, crude nuclear extracts could be used to
identify important sequences involved in VCP regulation. None of the above
techniques have been used in mosquitoes, and their development will
undoubtedly require effort. However, with the examples from Drosophila

available, such methods of analysis will certainly become common.

_ __.._-H

127
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