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This is to certify that the

thesis entitled
Identification and characterization of human

chromosome contents in somatic cell hybrids
from a patient with Menkes syndrome by using

G-ll stain technique

presented by

Shirong Wang

has been accepted towards fulfillment
of the requirements for

M . S . degree in Zoology

 

Date April 8, 1991

 

0-7 639 MS U is an Affirmative Action/Equal Opportunity Institution

 

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Michigan State
University

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TO AVOID FINES return on or before one due.

DATE DUE DATE DUE DATE DUE

      

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

MSU Is An Affirmative Action/Equal Opportunity Institution
cmmut

 

IDENTIFICATION AND CHARACTERIZATION OF
HUMAN CHROMOSOME CONTENTS IN SOMATIC CELL HYBRIDS
FROM A PATIENT WITH MENKES SYNDROME

BY USING G-ll STAIN TECHNIQUE

BY

Shirong Wang

A THESIS

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

MASTER OF SCIENCE

Department of Zoology

1991

ABSTRACT

IDENTIFICATION AND CHARACTERIZATION OF
HUMAN CHROMOSOME CONTENTS IN SOMATIC CELL HYBRIDS
FROM A PATIENT WITH MENKES SYNDROME

BY USING 6-11 STAIN TECHNIQUE

BY

Shirong Wang

A female patient carrying a de novo balanced
chromosome translocation t(X;2) (q133q32.2) has recently been
reported to fully express an X—linked recessive condition,
Menkes disease. This case is utilized to map the Menkes gene
on the human X chromosome. A modification of the sequential G-
11 banding procedure of Wyandt was employed to identify human
chromosome contents in four somatic cell hybrid clones
obtained from this patient. Differential staining appeared at
pH 11.97-12.05 and stain time 3-3.5 minutes. All cell lines
were found to contain either the intact or part of the human
derivative 2, including the breakpoint at Xq13. A detailed
discussion of the human chromosome contents in each clone, the
chromosomal analysis of the derivative 2 and inconsistency of

the 6-11 stain technique is presented.

To
My Parents

for their love and support

iii

ACKNOWLEDGEMENTS

I would like especially thank.my advisor Dr. James V.
Higgins for his guidance, support and encouragement during the
course of this experimental study.

I also thank Dr. Hackel and Dr. Champness for serving
on my committee and providing pointed comments.

I am grateful to Beverly Sankey for her technical
assistance and generously viewing the karyotypes.

Thanks are likewise due Dr. Glover, Vera Verga and
Bryan Hall for providing the specimens and for constructive
suggestion in performing G-ll staining.

Finally, I am indebted for the friendship of many
people in the Genetics lab who have made these years enjoyable
and memorable to me. They are: Julie Shreeve, Mickey Lopez,
Mecky Howell, Sirinunt Hope, Gillian Bice, Charlotte Wilks,
Lisa Stempek, Joseph Marrazza, Kim Brown, Blair Bullard, John
Bice, Rachel Eggleton, Paul Love, Donna Lehman, Linda
Kentfield and Alma Cantu. Thanks also to Mike Klusowski and
David Loomis for their technical assistance in word-processing

this thesis.

iv

TABLE

LIST OF TABLES . . . .
LIST OF FIGURES . . . .
INTRODUCTION . . . . .
LITERATURE REVIEW . . .

Menkes Syndrome .
Gene Mapping . .

OF

CONTENTS

Somatic Cell Hybridization

Human Cytogenetics

Differential Staining Techniques

MATERIALS AND METHODS
Culture . . . .
Harvest . . .
Making Slides .
Giemsa Banding
Destaining Sl des
Giemsa-ll Staining

RESULTS . .

Optimal pH of the Buffer
Human Chromosomal Composition in

Clones . . .

and

the

Staining Time

Four Cell Lines
Rearrangement of the Human Derivative 2 in Four

Cells Missing the Locus for the Menkes Gene .

Chinese Hamster Chromosome Numbers in Four Cell

Lines . . . .
DISCUSSION . . . . . .
SUMMARY . . . . . . . .
LIST OF REFERENCES . .

vi

vii

LIST OF TABLES

Table Page

1 Total Number of Cells Photographed after
Giemsa Banding . . . . . . . . . . . 28

2 Numbers of Slides and Cells Analyzed and

Usable after G-11 Staining . . . . . . . . . 29
3 Human Chromosomal Content in Four Clones . . . . . 31
4 Human Chromosome Composition in Different Cells . 33
S A Comparison of Different Individual Human

Chromosomes in Four Cell Lines . . . . . 34
6 Numbers of Hamster Chromosomes in Each Cell Line . 38

vi

LIST OF FIGURES

FIGURE
1 Blood karyotype of the patient C.G. . . . . . .
2 Chromosomes stained by G-ll technique . . . . . .
3a G-ll stained metaphase showing human chromosome
composition in MHCG 1A . . . . . . . .
3b Giemsa-banded metaphase showing human chromosome
composition in MHCG 1A . . . . . . . .
4a G-ll stained metaphase showing human chromosome
composition in MHCG 9 . . . . . . . . .
4b Giemsa-banded metaphase showing human chromosome
composition in MHCG 9 . . . . . .
5a G-ll stained metaphase showing the intact human
derivative 2 present in MHCG 10 . . . .
5b Giemsa-banded metaphase showing the intact human
der 2 present in MHCG 10 . . . . .
6a G-ll stained metaphase without the derivative 2
from MHCG 10 . . . . .
6b Giemsa-Banded Metaphase without the derivative 2
from MHCG 10 . . . . . . . . . . . . .
7a G-11 stained metaphase showing the translocated
der 2 in MHCG 10 . . . . . . . . . . .
7b Giemsa banded metaphase showing the translocated
der 2 in MHCG 10 . . . . . . . . . .
8a. G-ll stained.metaphase showing the translocated Xq
in MHCG 10 . . . . . . . . . . . . . .
8b Giemsa banded metaphase carrying the translocated
Xq in MHCG 10 . . . . . . . . . . . . .
9a G-ll stained metaphase showing human chromosome
composition in MHCG 11 . . . . . . . . .
9b Giemsa banded metaphase showing human chromosome
composition in MHCG ll . . . . . . . . .
10 Karyotype of MHCG 1A . . . . . .
11 Karyotype of MHCG 9 showing the translocated
derivative 2 . . . . . . .
12 Karyotype of MHCG 10 shewing the rearranged human
Xq . . . . . . . . . . . . . . . .
13 Karyotype of MHCG 10 . . . . .
14 Karyotype of MHCG 11 showing the translocated
derivative 2 . . . . . . . . . . . . . .
15 Karyotype of MHCG 10 . . . . . . . . . . . . .

vii

Page

49
50

51
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55
56
57
58
59
60
61
62
63

64
65

66

67
68

69
70

INTRODUCTION

Menkes Syndrome is an X-linked recessive disease.
Previous studies with comparative gene mapping on an animal
model of Menkes disease suggested that the possible locus for
this gene in humans is at band qu3 (Horn et a1, 1984). A
female patient with Menkes Syndrome was reported carrying a de
novo balanced translocation t(X;2) (ql3;q32.2). Her normal x
was found non-randomly inactivated, and presumably the
translocation disrupted the normal function of the Menkes gene
on the rearranged X, resulting in expression of the disease.
This case is being utilized at the University of Michigan to
map precisely the locus for Menkes gene in humans. The
lymphoblastoid from the patient C.G. was hybridized with the
Chinese hamster fibroblast cell line and HAS medium was used
to isolate the translocated human X:2 chromosome. The hybrid
cells from different clones were harvested and sequentially
banded with Giemsa and G-ll techniques to identify the human
DNA materials in these clones.

The G-11 technique is a Giemsa staining procedure.
conducted under pH of 11. It was primarily used to stain
selectively some heterochromatic regions in human chromosomes,
especially the secondary constriction of chromosome 1, 9 and

16, but was later found to be more valuable in differentiating

2

species-specific chromosomes in somatic cell hybrids and in
gene mapping (Bobrow, 1974; Burgerhout, 1975: Friend et al,
1976).

The purpose of this study is therefore utilization of
G-11 banding technique to identify and characterize human
chromosome contents in hybrid clones obtained from the patient
with Menkes Syndrome and to select cell lines containing the
rearranged human X32 chromosome for further mapping of the

Menkes gene.

LITERATURE REVIEW

1- margins:

In 1962, Menkes1 described an apparent x-linked
recessive disease found in a family of English-Irish descent.
Five boys were affected with peculiar hair (depigmented,
sparse and short), growth failure, severe psychomotor
retardation and focal or generalized seizures. Their failure
to grow brought these affected infants to medical attention at
the age of a few weeks and death occurred in the first or
second year of life. Their hair was examined microscopically
and showed twisting spirally, varying in diameter along the
length of the shaft.and fracturing at regular intervals. Focal
cerebral degeneration and cerebellar atrophy were reported
based on two autopsies. Although the disorder, named Menkes
Syndrome, was thought to be metabolic, no obvious plausible
cause could be discovered at that time. In 1972, Dankszland
his collaborators found that a type of Australian sheep with
copper deficiency produced abnormal wool similar to kinky hair
in Menkes disease. This discovery lead them to investigate the
copper metabolism in 7 patients with Menkes Syndrome. They
noted that all of these patients had low serum copper and
ceruloplasmin, as well as abnormal intestinal absorption of

orally administered copper salt. In the paper published in

4
1972, they suggested that Menkes Syndrome was in fact an
expression of defective copper metabolism. Besides the low
serum copper and ceruloplasmin levels in Menkes disease, liver
copper levels were found to be grossly reduced, and copper
content in brain was also decreased. All other organs and
tissues showed an excessive amount of copper, especially in
the kidney which had the highest level of copper of all the
tissues. Copper absorption from the intestine has been
reported to be only 15% of normal (Dekaban et al‘, 1975). The
renal copper loss was excessively great. Studies on cultured
fibroblast cells from the skin of patients demonstrated the
accumulation of excessive amounts of copper and the increased
sensitivity to the toxic effects of additional copper added to
the culture medium (Danks‘, 1977). Based on these
observations, Danks (1977) and William5 (1978) postulated that
the basic defect in Menkes Syndrome resided on a mutation of
an intracellular copper-binding molecule causing an increased
binding affinity to copper. As a result, copper enzyme
synthesis would be impaired despite a normal level of cellular
copper. This binding molecule, named metallothionein, was
found to be present in increased amounts in most tissues.
However, this earlier hypothesis has been discarded when
evidence showed the location of the gene for metallothionein
to be on chromosome 8 in mice and chromosome 16 in humans, and
not on the X chromosome (Danks‘, 1989) . In recent years,

metallothionein cDNAs has been cloned from several species and

5

the expression of this gene has been well-studied. Measurement
of its mRNA indicates that the increased induction of the gene
is due to increasing levels of copper rather than the pre-
existing overexpression of the genes (Danks, 1989) . The
explanation for this finding is that a gene encoding for an
intracellular copper transport protein has mutated and this
abnormal protein loses its function of carrying copper to the
intracellular sites where copper enzymes are assembled. As a
result, excessive "free" copper induces metallothionein
synthesis. It is apparent that, instead of being a disease of
simple copper deficiency, Menkes Syndrome is a storage disease
in which copper is trapped by metallothionein in several
tissues, most notably in the kidney. The resultant deficiency
of copper in other tissues and the impairment of the
biosynthesis of copper enzymes accounts for the generalized
defects. The incidence of the Menkes disease is estimated 1 in
50,000 to 100,000 (Danks et a1, 1989).

In 1987, Kapur et al7 reported a female patient with
Menkes disease having a de novo balanced translocation between
chromosomes X and 2. The patient C.G. was an 11 month old baby
born to a 28-year-old G2, P1 mother and an 33-year-old father.
She was delivered by Cesarean section at 35 weeks. Her birth
weight was 2,610 gram, length 48.5 cm, and head circumference
35 cm. She was normal at birth and discharged from the
hospital at the normal time. Starting from 6 months she

developed seizures and had feeding difficulty as well as

6

growth retardation. Her hair was scanty with a visible fine
kink. Microscopical examination of the hair showed typical
pili torti. Serum copper was as low as 14 ug/dl compared to
the normal level of 70-150 ug/dl and serum ceruloplasmin 8
mg/dl (normal 18-45 mg/dl). The peripheral blood chromosomes
were studied and karyotype was reported as 46,X,t(X;2)
(q133q32.2) at 550 band level (Figure 1). Her blood showed
late replication of the normal X chromosome in.all 24 spreads.
This result indicated that the normal X chromosome ‘was
inactivated in this patient. Both her parents had a normal
karyotype. Since Menkes Syndrome is an X-linked recessive
disease, females are usually not affected. It was therefore
hypothesized that non-random inactivation of the normal X
chromosome resulted in the expression of the defective Menkes
gene on her active X translocated chromosome.

There are similar case reports where a known X-linked
recessive condition is fully expressed in females bearing an
X-autosomal translocations. In his review article, Mattei8
analyzed 105 cases with translocations between X and different
autosomes. He found that in nearly 95% of balanced t(X-
Autosome) and 91% of unbalanced t(X-Autosome), X chromosome
inactivation occurred in the most favorable model rather than
as a random event (Lyon’ 1961) . In the case of a balanced
translocation, inactivation of the translocated X will lead to
spreading of the inactivation into the adjoining autosomal

material, thus creating a partial autosomal monosomic

7
condition. Therefore, the normal X is seen to nonrandomly
inactivate. The converse is true in an unbalanced
translocation in which spreading of the inactivation into the
partially trisomic or monosomic autosome confers a selective
advantage, and the translocated X will be nonrandomly
inactivated. Mattei concluded that. most females with a
balanced X/autosome translocation showed preferential
inactivation of the normal X. This would allow for the full
expression of a recessive allele on the translocated X,
whether it was caused by an inherited gene mutation or by the
chromosome rearrangement. The same conclusion was also made by

Gartler and Sparkes1o (1963).

2. Mains:

There are many different ways to map a gene. From the
cytogenetic point of view, a powerful method to map a gene to
a specific band of the chromosome is the utilization of a
balanced X/autosome translocation in female patients with full
expression of X-linked recessive diseases. Duchenne muscular
dystrophy (D.M.D) is an example. Boyd et al11 (1986) reviewed
20 cases of female patients carrying X-autosome
translocations. These translocations involved different
autosomes but the breakpoints on X chromosome were all in the
short arm at band 21, without exception. Presumably the
rearrangement at this specific site had disrupted the normal

function of the gene for D.M.D and this, coupled with the

8

preferential inactivation of the normal X chromosome, was
responsible for the occurrence of the disease. It would be
reasonable to hypothesize therefore that the chromosome
breakage was at the gene locus for D.M.D., and had created a
mutation and subsequent full expression of phenotypes due to
non-random inactivation of the normal X chromosome. The
assignment to Xp21 has been confirmed by family studies using
the probes which detect restriction fragment length
polymorphisms of the X chromosome short arm (Goodfellow et
a1”, 1985) . Theoretically, the gene for Menkes could also be
mapped in the same way by using translocation as a tool, but
practically it is not feasible since Menkes disease is a
relatively rare disorder and no second case has been reported
to carry a translocation between the chromosome X and an
autosome.

Nevertheless, some advances have been made in recent
years in mapping the human gene for Menkes Syndrome by using
DNA hybridization technique. Wieacker13 (1983) performed a
linkage study in a large kindred with Menkes Syndrome by using
RFLP probe 1.28, which mapped to the distal part of Xp between
X centromere and Xp11.3. He found at least two crossovers. The
distance was estimated at 18 cM. Other data using different
RFLPs probes, such as MGUZZ (close to the centromere) and a
centromeric C-banding polymorphism also indicated the linkage
of Menkes gene to the centromeric region (Ropers et a1“, 1983;

Friedrich et a1“, 1983). Wienker et all“ (1983) assigned

9
Menkes gene on the short arm of X and proposed the most likely
gene order as Xpter-Menkes-Ll. 28-MGU22 . However, a comparative
mapping conducted by Horn et al"'(1984) suggested that the
Menkes locus was likely on the long arm close to band q13.
This conclusion was reached based on an animal model of Menkes
'disease in the mouse.

The Mottled.mice (an animal model of Menkes Syndrome)
were initially noted by their variegated depigmentation of the
coat.color in females heterozygous for this X-linked.gene. The
deficiency of male offspring suggested lethality of this
disorder in male. A number of allelic mutations have been
recognized in mouse. The males with less severe mutations had
severe depigmentation, abnormal vibrissae, skeletal defects,
neurological degeneration and arterial abnormalities
(Philips”, 1961; Rowe”, 1974). The mutation in the mottled
mouse is considered to be homologous to Menkes Syndrome in the
human being with the same pattern of inheritance and similarly
neuropathologic, skeletal, vascular, and clinical
abnormalities, as well as similar copper abnormalities in
tissue and serum and abnormalities in copper metabolism
(Danks, 1977). X-linkage of the Mo locus (‘mottled') in the
mouse has been proven and the locus has been mapped on the X
chromosome of the mouse near the loci for phosphoglycerate
kinase (ng-l) andnalphagalactosidase (Ags) and.closely linked
to ng-l (McKusich, 1978). Since the order of ng-l/ng and

Ags/Gal in mice and humans is the same with respect to the

10
centromere (Horn et a1, 1984) and the human PGK has previously
been mapped to band Xq13.2-13.3, the most likely position of
the gene for Menkes in humans is on the long arm of X

chromosome, within the band qu3.

3- MW:

Somatic cell hybridization was begun in 1960's as a
product of the development in somatic cell genetics. As early
as 1961, Barski et alz1 first noted proliferating cells arising
from fusion of the two lines which contain the chromosomes of
both cell lines. These results were confirmed by others, and
Littlefieldni(1964) added a selective system, hypoxanthine-
aminopterin-thymidine (HAT), to isolate hybrids between the
two drug-resistant cell lines. Ephrussi et ala’(1965) were
able to fuse human-rodent hybrids and isolate them in a HAT
selective medium. The medium contains hypoxanthine and
thymidine (H and T), the normal precursors of DNA, and
aminopterin (A), a drug that specifically blocks one pathway
leading to DNA synthesis. Normal cells can synthesize DNA by
way of two metabolic pathways: the major pathway, which is
blocked by aminopterin, and a salvage pathway which uses
nucleoside as a precursor to synthesize DNA. Two enzymes are
required for completion of the salvage pathway: thymidine
kinase (TX) and hypoxanthine-guanine phosphoribosyl
transferase (HPRT) . Cells that are unable to produce either TX

or HPRT cannot convert precursors into DNA along the salvage

11

pathway and must depend on the major pathway for DNA
synthesis. Thus, if the major pathway is blocked by the
presence of aminopterin in the medium, such cells would be
unable to grow. Grzeschik et al“’(1972) made hybridization
between a human cell strain derived from a female heterozygous
for an X-autosome translocation in which the structurally
abnormal X was genetically active and a HPRT deficient mouse
cell line which could not survive in HAT medium and therefore
required the human HPRT enzyme to proliferate. In these
hybrids, cells underwent progressive reduction in the number
of human chromosomes, but selective retention of the human X
chromosome on which the HPRT gene is located. By this way, the
reduced hybrids containing only one of the two segments of the
structurally abnormal X chromosome can be obtained. These
kinds of cell clones are very useful to map a specific gene on
the X chromosome. The DNA samples from these clones and from
the patient's family members can be digested with the
appropriate restriction endonucleases and analyzed for RFLPs
by Southern blot.hybridization with selected radiolabeled DNA
probes.

Human lymphocytes are often used for somatic cell
hybridization, but its limited lifetime prevented its use in
long-term culture. Usually at about 40-45 days it will lose
viability and die. Gerrer et al25 (1969) in vitro infected
buffy-coat cells from the peripheral blood of a healthy adult

with a herpes-like virus designated Epstein-Barr virus (EBV)

12
and successfully established a long-term proliferated
lymphoblast culture. In this culture, seventy-five percent of
the cells which had been transformed by a virus showed
striking changes in morphology: lymphocytes grew in size from
small cells to medium and large ones with sparse cytoplasms
and the enlarged rounded nuclei contained prominent nucleoli.
These transformed cells obtained much extended life span,
therefore could be used for the establishment of long-term

cell clones for DNA analysis.

4- W:

The study of human chromosomes can be said to date
from 1879 when Arnold observed chromosomes in dividing human
cells (Makinoz‘, 1975) . In the following years, many scientists
attempted to determine chromosome numbers in humans. However,
the correct number was not established until 1956 when Tjio
and Levanz7 reported consistent counts of 46 chromosomes in
fibroblast cultures from the lungs of human abortuses. This
discovery was attributed to technical improvements in
cytogenetics in the 19505 which allowed for the accurate and
precise examination of chromosomes.

One of these achievements was obtaining satisfactory
preparations of dividing cells at metaphase. For the majority
of the cell cycle time, the chromosomes are in an interphase
in which the DNA is diffuse and no details of the chromosome

can be seen. Whereas during metaphase, the DNA is maximally

13
condensed and has distinctive morphology. However, only a
small proportion of cells are in metaphase at any one time.
Even though metaphase cells can be observed, the clumping and
overlapping of chromosomes always make results uncertain. In
the 1950s, several improvements in technical methods were
obtained in the following fields:

The first of these was the development of techniques
for culturing tissues and cells in vitro. Classical studies of
human chromosomes were limited to testicular specimens which
had the problem of the relative scanty number of cells
undergoing division. By systematic addition of the various
nutrients required for mammalian cells, people were able to
culture in vitro a variety of tissues. Newly developed
techniques involved the use of short-term cell cultures. These
cultures furnish actively dividing cells without a loss or
gain of chromosomes. A higher proportion of mitoses could be
obtained through cell harvest from these short-term cultures.

A second technical development was the application of
colchicine or its derivative Colcemid on tissue culture. This
compound interferes with the formation of the mitotic spindle
so that when a cell enters mitosis under the influence of
colchicine, the spindle fails to form and the cell is
therefore "arrested" at metaphase (Levanza, 1938) . Thus,
colchicine treatment increases the opportunity of finding
cells suitable for examination. The use of Colcemid can

eliminate some toxic side effects of colchicine such as a

14
chromosomal contraction.

A third development was the treatment of cells with
hypotonic solutions of various kinds for the purpose of
swelling cells in order to improve the chromosome dispersion.
Makino and Nishimura”’(1952) had used water pretreatment of
cells for the study of the chromosomes in animals. Hsu3° (1952)
developed a method of using hypotonic culture medium on human
fibroblasts. The chromosomes examined by this method appeared
as sharply defined bodies with considerable spreading in the
cells, making them more amenable for study. There was minimum
overlapping in well-prepared metaphase and the chromosomes
could be counted readily by means of photomicrography. Other
various solutions used included hypotonic sodium citrate and
sodium chloride, 0.65M potassium glycerophosphate, 0.075M
potassium chloride, and diluted balanced salt solutions of
varying complexity (Hanks, Ringer, Tyrode, etc).

Improvements were also made on slide preparations. The
previous method introduced by Hietz in 1936 was called "the
squash technique" (reviewed by Hamerton”, 1971). It involved
putting the materials between a coverslip and a slide, and
with gentle but firm pressure, pressing the material until the
chromosomes spread. One problem with squash preparations,
however, was that they were temporary and slowly deteriorated.
Also, mammalian cells were very soft after hypotonic solution
pretreatment and pressing too hard would break cells.

Therefore, when "air-drying technique" was developed by

15

Rothfels and Siminovitch32 (1958), it became a welcome
technical improvement and quickly replaced the previous
”squash method" . ”Air-drying technique" basically consisted of
placing a drop of cell suspension on a clean slide, allowing
it to spread out, and then drying quickly. This method gave
good reproducible results with a high yield of well-spread
metaphase chromosomes. Since then variations were made on this
technique, such as the method developed by Moorhead” (1960)
who used a methanol-acetic acid mixture as fixative to force
chromosomes spreading without any mechanical pressure.
Methanol-acetic acid has a property of quick evaporation. It
takes water out of cells quickly so that cells are able to
flatten out and the chromosomes will spread.

Another major revolutionary breakthrough in
cytogenetic technology was the discovery of various banding
methods which allow the accurate identification of individual
human chromosomes. Eighty years ago, a German scientist,
Gustav Giemsa, found that a degradation product of methylene
blue, called methylene azure, was able to stain chromatin and
that another compound, eosin, contributed to it by binding the
azure-incorporated chromatin. He then mixed methylene azure,
eosin, glycerine and methanol together to form a stain
solution, later named Giemsa stain (reviewed by Hsu“; 1979).
Prior to that time cytologists often used acetic orcein,
acetic carmine and other stains, rather than Giemsa, for

chromosome preparations. Those stains had certain limitations

16
in the identification of the human chromosomes on
morphological criteria , and consequently structural
rearrangements occurring in the chromosome segment will often
escape detection. Thus, there was a need for more objective
methods that would allow the identification of individual
chromosomes with accuracy. In response to this demand, several
banding techniques were developed in late 60's and early 70's:
Caspersson et a135 (1969) discovered Q-band, quinacrine
fluorescent banding, using quinacrine mustard. He found
characteristic fluorescent segments (or bands) of various
degrees of brightness along each metaphase chromosome. Thus
chromosome pairs with identical gross morphology could be
recognized individually by their fluorescence banding
patterns: Arrighi et al"“5 (1971) established C-band (a term for
constitutive heterochromatin) technique by which
heterochromatic (including centromeric) regions showed darker
Giemsa stain than the chromosome arms: By applying the C-band
procedure to flame-dried slides, Patil et al"‘7 ( 1971) was able
to find a new banding pattern, later named as G-band (Giemsa-
band). A comparison was made between this new Giemsa banding
and Q-banding in the Paris Conference that fall. The two
systems matched almost band by band, with the brightly
fluorescent Q-bands being equivalent to deeply stained G-
bands, and the dull or non-fluorescent Q-bands corresponding
to the lightly stained or unstained G-bands. In the same year,

Seabright""B modified the Giemsa technique by pretreatment of

17
the mitotic spreads with trypsin and obtained sharp
chromosomal banding patterns. This trypsin Giemsa technique
has replaced the older Giemsa—band methods and has become a
routine chromosomal banding technique used in most cytogenetic
laboratories since. Simultaneously with the discovery of G-
band, Dutrillaux found R-band (Reverse Giemsa band) which is
exactly the reverse of G-band in the sense of the darkly and
lightly stained zones (reviewed by Hsu, 1979) . As demonstrated
with the Q-, G-, R-or C-staining methods, each individual
chromosome appears as consisting of a continuous series of
light and dark bands organized in a well defined differential
patterns. All of these banding techniques gave a similar
staining pattern of the chromosomes, therefore they provided
a useful and reliable tool for identifying each chromosome by
recognizable characteristic band patterns produced along the

chromosomes.

5. 2W:

To map human gene, a very useful technique is to
produce interspecific hybrid via somatic cell hybridization.
Such crosses often undergo an initial random loss of human
chromosomes until karyotype stability is reached. By
comparison of the cosegregation of human gene products in a
number of hybrid clones, linkage of the gene loci determining
those products can be inferred. This technique requires

distinguishing and identifying human genetic material from a

18
rodent's background so that a gene locus could be assigned to
a given human chromosome. A variety of techniques have so far
been reported to obtain staining differences between different
species. Two such methods are Hoechst 33258 staining and C-
banding techniques, which offer a rapid means of
distinguishing human and mouse chromosomes.

In 1972, Hilwig et a1” conducted a test to treat
chromosome preparations from ‘mouse cells with the
bibenzimidazlo compound "Hoechst 33258" (H 33258). He found
intense staining of the centromeric regions on the mouse
chromosome. Kucherlapati et al‘° (1975) applied the same
procedure on mouse-human somatic cell hybrid and observed a
differential staining between mouse and human chromosomes.
With this stain, the mouse chromosomes in the hybrid cells
showed bright fluorescence whereas the human chromosomes were
non-fluorescent or weakly fluorescent.

The C-band method developed by Arrighi and Hsu (1971)
uniquely stains heterochromatin regions and is also used to
identify specific human and mouse homologous chromosomes. In
the hybrids stained with C-band technique, the mouse
chromosomes had a deeply staining block of heterochromatin
which accounts for approximately 10% of the total length in
the longer chromosomes and up to 50% of the total length in
the shorter chromosomes. In contrast, the centromeres of human
chromosomes were not stained as intensely as those of mouse

chromosomes because constitutive centromeric heterochromatin

19

in man is quantitatively reduced compared with the mouse (Chen
et a1“, 1971).

Although C-banding technique and staining with Hoechst
33258 could provide a rapid means of identifying mouse and
human chromosomes, they usually fail to detect parts of and
small translocations of the human chromosomes. In 1972, Bobrow
et al‘2 reported a new Giemsa stain technique, named G-11
staining. The basic mechanism is that under highly alkaline
conditions (Ph=11), Giemsa mixtures can differentially stain
certain heterochromatic regions into magenta and remaining
portion of human chromosomes pale blue. It was originally
adopted for use in cytogenetic analyses of patients with
hematologic conditions, primarily leukemia. It was also used
to demonstrate polymorphisms, translocations and pericentric
inversions. Modifications of this procedure have been useful
for differentiating species—specific origin of a chromosome in
human-rodent hybrid cells (Bobrow et a1", 1974) . Under optimal
conditions, mouse chromosomes in the hybrids stain a dark
magenta with pale blue centromeres whereas human chromosomes
show blue color with red differentiation at paracentromeric
regions. By using Giemsa-11 technique, Burgerhout“’(1975) and
Friend“5 (1976) were able to successfully detect two-colored
chromosomes‘which represented interspecific translocations in
the mouse and human hybrids. Such an identification would be
very helpful to aid in the regional localization of human

genes.

MATERIALS AND METHODS

1. culture:

The somatic cell hybrids between lymphoblastoid cells
from the patient C.G. and Chinese hamster fibroblast cell line
RJK88 was constructed in the genetics laboratory of the
University of Michigan. Fusions were performed using
polyethylene glycol (PEG) and hybrids were selected in HAS
medium (hypoxanthine/ azaserine) for the retention of the
derivative chromosome 2 (2pter-2q32:Xq13-ther). Fourteen
hybrids were isolated and four of them were obtained from the
University of Michigan for further identification by G-banding
and G-ll staining. These frozen cell cultures were thawed by
removal of the freezing ampoules from the styrofoam box
containing dried ice and. immediate immersion. in a 37'C
waterbath, agitating until defrosted. Cells were then
transferred into the 25 cm? Falcon flasks with the Pasteur
pipettes, suspended in 5 ml HAS growth medium immediately, and
finally incubated at 37'C in a 5% CO2 incubator. HAS medium
was made from 500 ml D-MEM supplemented by 10% fetal calf
serum, 2% L-glutamine, 10 ml 104 M L-proline, 20 ml 2.5x105
M hypoxanthine, 5 ml 10'3 M azoserine and 10 ml
Penicillin/streptomycin.

Cultures were inspected under the microscope for the

20

21
growing density every other day. If it reached 1/2 confluence,
cells would be subcultured into another flask. To do this,
cells were first rinsed with 1X PBS, then exposed to warmed
(37'C) 1.0 ml 0.25% GIBCO trypsin-EDTA for 1 minute or until
most cells were round up and detached from the flasks. 0.7 ml
cell suspension was discarded from each flask, followed by
addition of 5 ml complete HAS medium and mixed. When cultures
showed healthy growth with 1/2 confluence or less, enzymatic
disposal was not necessary: instead, a change of fresh medium
would be sufficient. If individual cultures were growing
poorly, only half of medium was discarded from the flask each
time and replaced by an equal amount of fresh HAS medium. By
microscopic monitoring, cultures were allowed to be in the

active growth condition until they were ready for harvest.

2- harxest:

Harvest was usually accomplished when cells were 2/3
confluent. Twelve hours before harvest, cultures were fed with
the fresh HAS medium in order to synchronize the cell cycle.
Just before the harvest, they were washed with 1X PBS followed
by 1 ml GIBCO Trypsin-EDTA digestion for 1-2 minutes. Free
cells were transferred to centrifuge tubes and treated with
colcemid at final concentration 0.1 ug/ml for 20 minutes to
accumulate metaphase cells. Cultures were then removed from
incubation and spun for 5 minutes at 1000 RPM in a centrifuge.

After the supernatant was drawn down to 1 ml, pellets were

22
tapped gently to mix well. Cultures were then treated with 7
ml prewarmed (37'C) 0.075 M KCl and allowed to incubate for 12
minutes. Timing is the critical step in minimizing the amount
of cytoplasmic background in order to obtain good color
differentiation. Hypotonic treatment for 12 minutes in 0.075M
KCl is sufficient to achieve a minimum background. At the end
of 12 minutes, 1 ml freshly-made fix (3:1 100% methanol and
acetic acid) was added directly into hypotonic solution and
mixed well. Then the suspension was centrifuged at 1000 RPM
for 10 minutes. After supernatant was removed, cultures were
treated with 8 ml 3:1 fix and allowed to be refrigerated at
4'C for 10 more minutes. Two changes of fix were required
before the completion of harvest. At the end, pellets were

stored at 4'C for the future use.

3. Wes: '

The pellet was taken out from the refrigerator and
washed once or twice with fresh fixative. The supernatant was
drawn down to the appropriate level and cell suspension was
gently mixed by a Pasteur pipette. The slides were precleaned
with 100% ethanol and. then rinsed ‘thoroughly «in. double
distilled water. Spreading of the metaphase preparation was
achieved by pipetting three drops of low density cell
suspension to separate areas of each wet slide from 10-15"
height. The slides were then passed through a steamer for

about 2-3 seconds and placed on a 65'C slide warmer for 1-2

23
minutes or until the water was removed. Slides on each cell
clone were scanned to determine their quality on a low power
lens using a phase contrast scope. The methods utilized were
adjusted to obtain appropriate cell density, high mitotic
index, optimal chromosome spreading and minimal cytoplasmic
background. Adjustments could be made by increasing or
decreasing the angle of slide, dropping cell suspension from
more or less height, passing slides through a steamer for
longer or shorter time, blowing over slides or avoiding
blowing, as well as changing the ratio of methanol:acetic acid
in the fix, etc. Once the optimal method was determined, all
other slides on the same clone would be made in the exactly
same way to ensure the consistency of each slide. The slides
were then aged in 90'C oven for 1 hour and 15 minutes and
stored.in an air tight container. The critical point in making
slides was toikeepicell density low. Excess cells would result
in an overall blue appearance of chromosomes after staining.
The density was thought to be appropriate if cells spread over

1/3 to 1/2 area of a slide.

4- MW:

Trypsin stock solution was made by dissolving 2.5 g
SIGMA type III Trypsin powder in 100 ml Tyrode solution which
contained NaCl 8.0 9, KCl 0.2 g, NaHZPO,°HZO 0.05 g, Glucose
1.0 g, NaHCO3 1.0 g and 1000 ml distilled water. This Trypsin

solution was then aliguoted into Falcon tubes, 1 ml in each,

24
and stored at 0’0. Before banding, one tube was removed from
the freezer and thawed out under room temperature. 0. 3 ml
Trypsin stock solution was diluted with 100 ml 1X PBS, forming
Trypsin working solution.

To make the Wright stain solution, 2.5 g of EM Wright
blood stain powder was mixed with 1000 ml methanol, stirred
for four hours and then filtered through two combined #1
filters.

Slides were trypsinized for 10-20 seconds, rinsed
thoroughly with two changes of 0.9% NaCl and blotted. They
were then stained for 1 minute 10 seconds to 1 minute 20
seconds with 4 ml Wright working solution (1 ml Wright stain
solution + 3 ml Gurr's buffer-pH 6.8). When the staining was
complete, the slides were rinsed with double distilled water,
then air dried and examined for banding quality by using 100x
bright field oil immersion lens: if chromosomes in most
mitoses appeared bloated, empty or fuzzy, over-trypsinization
was suggested and the next slide would be trypsinized for a
shorter time: if chromosomes were under-trypsinized as
indicated by decreased contrast and indistinct bands, exposure
time to the trypsin would be increased. Staining time was also
adjusted by monitoring color intensity of chromosomes.

Stained slides were scanned on low power bright-field
objective to select well-banded, well spread mitoses, with as
few overlapping chromosomes as possible. These metaphases were

further examined on 100X bright field oil immersion lens and

25
photographed with 35 mm high contrast copy films. The co-

ordinates of the scope was written for the record.

5. W:

1 After pictures were taken, slides were cleaned with
xylene. The destain process involved dipping these slides
sequentially in 70% ethanol, 80% ethanol, 95% ethanol + 2%
hydrochloric acid, 95% ethanol and 100% methanol, and finally
blotting dry.

6. We:

The method used was modified from Wyandt et al (1976) .
To make G-ll working solution, CAPS buffer and G-11 stock
solution were needed. The buffer was made by dissolving 2.213
gram SIGMA CAPS powder in 1000 ml distilled water to obtain
the final concentration 0.01 M. CAPS was warmed in 37'C water
bath for 1 hour. Before making G-11 working solution, the pH
of the buffer was adjusted to the desired value with a pH
meter.

Giemsa-11 stain solution was made by combining 0.5
grams powdered Giemsa (Fisher G-146) and 33 ml glycerol,
stirring for 2 hours followed by incubation overnight at 37'C.
On the second day, 33 ml methanol was added and thoroughly
mixed.

The destained slides were placed in a Coplin jar and

soaked in distilled water for 1-2 hours. Just before staining,

26
they were transferred directly from distilled water to 49 ml
prewarmed (37°C) and pH pre-adjusted CAPS buffer in water
bath. Immediately 0.8-1 ml Giemsa-11 stain solution was added
into the buffer and mixed gently to minimize formation of
surface film which would adhere to a slide during its removal
from the solution. Staining of the slide was monitored by
removing them at various time intervals and reviewing
microscopically for color intensity. If the chromosomes
appeared pale blue overall, the slides would be stained
longer: if the chromosomes were very dark magenta, the
staining time would be reduced. The average time was 3 - 3.5
minutes for all the slides. At the end of the staining time,
slides were removed, rinsed briefly in distilled water, dried
with compressed air and mounted with cover slips. The pre-
photographed slides were viewed again under the same
microscope and those metaphases formerly photographed were
relocated with the previous co-ordinater record. If color
differentiation occurred on these mitoses, a second photograph
was obtained by using either colored or black/white film and
two prints of the same spread were compared to determine the
human chromosomal contents. The stain solution was always
prepared fresh for each set of 2-3 slides and could not be

reused.

RESULTS

With well stained slides, human chromosomes are light
blue with red dots at paracentromeric region of chromosomes 1,
4, 5, 7, 9, 10, 13, 14, 15, 20, 21, and 22. Chinese hamster
chromosomes are stained a dark magenta, with pale blue
centromere region (Bobrow, 1974). In hybrids, human X
chromosome can be identified by its lighter color and by the
absence of the markedly paler centromere region. Occasionally
a translocation can occur between human and Chinese hamster
chromosomes. Under'these conditions, translocated chromosomes
will be stained blue and magenta respectively (Figure 2): in
a black-white photograph, the respective areas are displayed

as light and dark gray.

1- WWW:

Four cell lines were tested, MHCG 1A, MHCG 9, MHCG 10
and MHCG 11. For each of them, a minimum 10 slides were made
to ensure that at least three slides would be able to show
differentiation after G-11 staining.

The starting pH of CAPS buffer was 11.50, at which all
the chromosomes were stained pink without any color
differentiation. This indicated the pH of CAPS solution was

too low. The pH was then raised by 0.1 each experiment and

27

28
slides were monitored microscopically to direct the
adjustment. The optimal differentiation was achieved at the
staining time of 3 - 3.5 minutes and pH of the buffer in the
range of 11.97-12.05. At this pH, all four cell lines showed
distinct color differentiation with human chromosomes stained
light blue and Chinese hamster chromosomes dark magenta. The
same differential staining was also found on the translocated
chromosomes. The blue areas of these chromosomes coincided
with the parts which were identified as being human-derived on

regular Giemsa-banded slides.

Table 1 Total Number of Cells Photographed After G-banding

 

 

 

 

 

 

 

 

 

 

 

I_ I an
cell lines # of slides # of cells
MHCG 1A 5 86
fl MHCG 9 9 81
II
MHCG 10 4 109 1
MHCG 11 8 135
Total 26 411
%—

 

 

As shown in Table 1, a total 26 slides and 411 cells

29

'l

 

 

 

 

 

  
 

 

    

 

 

 

 

 

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omeaamcc maaoo

 

mo mom «a 44509
ha Nm n Ha 00::
mm or n OH 00m:
n mm m m 60::
an «N mm m (a 00::
uaaoo sofiuoducououuwo oonaaoce couanoso
House: no a nu“: mauoo u0 n uuaoo no ¢ noowam mo e 0:«a Hamo

Uco mwcwam HO mumnaaz N wanna

30
were photographed after Giemsa banding. Table 2 lists the
number of cells with differentiation after G-11 staining. The
total number of cells photographed was 411, however only 68 of
them showed color differentiation. Among these 68 cells, 2/3
(46/68 cells) had distinctively blue and pink colors on the
human and Chinese hamster chromosomes, respectively.

In Table 2, the column " # of slides analyzed" refers
to the slides in which color differentiation was evident. It
does not include those in which differential staining was
absent in the previously photographed cells, even though
present in some other metaphases in the same slide. "The # of
cells analyzed" specifically indicates the chromosome spreads
which had already been photographed after Giemsa banding. "The
# of cells with differentiation" was counted from the analyzed
cells only, as described above. Again, the color
differentiation could also be found in other cells in the same

slides, but they were unable to be utilized because of the

lack of the sequential G-11 staining.

 

Although the number and content of human chromosomes
varied from cell line to cell line, the derivative 2 was found
in all four clones in either intact or translocated form. Cell
lines MHCG 1A, MHCG 9 and MHCG 11 did not bear the detectable
intact nor translocated normal or derivative X, based on

karyotyping analysis. The exception was MHCG 10, which will be

31
discussed later; MHCG 1A, 9 and 10 also carried. human
chromosomes other than the derivative 2, as seen in Table 3.
Table 4 and 5 give details of the human chromosomal contents

in each clone.

Table 3 Human Chromosomal Content in Four Clones

 

 

 

 

 

 

_——
# of cells presence of additional human
cell lines
analyzed der(2) chromosomes
MHCG 1A 22 yes 8, 12, 14, 22
MHCG 9 4 yes 14
MHCG 10 19 yes 8,13,22
MHCG 11 9 yes NONE
TOTAL 54 8,12,13,14,22
a“ Mm

 

 

 

 

 

 

 

MHCG 1A: Selected by HAS medium, the derivative 2 was
present in 18 out of 22 spreads, and found to be missing in
the other four cells. Besides the derivative 2, each spread
carried either one, two, three or all four of the following
human chromosomes—-8, 12, 14, and 22. These four chromosomes,

together with the derivative 2, consisted of the basic

32

component of the human chromosome in MHCG 1A cell line (Figure
3). Among the 22 spreads analyzed, 1 had all 5 of these
chromosomes, 7 had 4, 11 cells had 3 of them, 1 cell had 2 and
the remaining 2 cells had only one chromosome of human origin.
Thus the majority of cells (18/22) contained 3-4 human
chromosomes, which probably represented the stable number of
human chromosomes in this specific clone.

Three cells in MHCG 1A were marked with " ? " in Table
4. One of them had distinct color differentiation on all
chromosomes except one, which was stained between blue and
magenta on the short arm and dark magenta on long arm. When
reviewing its Giemsa-banded karyotype, the short arm of this
chromosome failed to match any part of human chromosomes.
~Since the color alteration was not distinguished on this
specific chromosome, it is likely that this segment of
chromosome is not human-derived. The other two questionable
cells containing the same piece of chromosome in light blue
color with two positive bands and without apparent centromere
visible in Giemsa banded karyotype. From the color and
morphology, it could represent a part of a broken human
chromosome. But since these pieces of chromosomes were small,
it was not possible to tell, by their morphology, from which
chromosome they had derived.

MHCG 9: Four cells were analyzed. The long arm of the
derivative 2 was again found in the majority of the cells

(3/4), human 14 was also present in 3/4 cells. These were

33

Table 4 Human Chromosome Composition in Different Cells

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

.--___. ......._______.
cell lines human chromosomal # of
composition/cell cells
22 1
14 l
14, 22 l
22, t(der 2g), ? 1
14, 22, t(der 2q) 8!
MHCG 1A
12, 14, 22 1
12, 14, 22, t(der 2g) 5
12, 14, t(der 2g) 1
14, 22, t(der 2g), ? 2
8, 12, 14, 22, t(der 2g) 1
14 1
MHCG 9 t(der 2q) 1 4
14, t(der 2g) 2
8 4
t(der 2g) 1
der 2 l
MHCG 10 13, der 2 l 19
8, t(Xq) 4
22, der 2 5
8, t(der 2q) 3
9

 

 

 

 

 

34

 

 

 

 

 

  

 

  

 

 

 

 

 

 

 

 

 

 

    

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8 m~\m a -\H o
as 00:: as cos: a com: 4” cos:

amass:

O: 00 DOT EOH m 00 O a O OEOmOEOH O
. a” as a u as u . H u u \ n msomosouso

sees: ofiuuooom a mcwueon uHHmo no u v canon

 

q

 

mesa; Haoo noon :« nesomosouzu sees: dosow>wosn acououuflo uo conflueosoo 4 m wanna

35

the only two human chromosomes retained in MHCG 9 (Figure 4).

MHCG 10: The chromosomes of human origin were the
derivative 2 (intact and part) in 11 out of 19 cells, the
translocated Xq in 4 cells, chromosome 8 in 11 cells,
chromosome 13 in 1 cell and chromosome 22 in 5 cells (Figures
s-s).

MHCG 11: Nine cells were examined in this cell line.
All of them contained a single chromosome which had both human
and hamster staining materials as showed in Figure 9. It was
identified to be a translocation between the human derivative
2 and. a Chinese hamster chromosome. This ‘was the only
chromosome containing human genetic material detected.by G-11

staining in MHCG 11 clone.

3. ;-e 20°‘u‘r .- ,- o.ue! .- a - I -_ 018‘:

As shown in Table 5, the derivative 2 was found in 41
out of total 54 spreads from all four clones. In 34 spreads it
was translocated to different Chinese hamster chromosomes
(Figures 10, 11, 12, 13 and 14) while in seven cells, it was
found intact.

The break points of the derivative 2 were also
different from cell line to cell line. In MHCG 1A and MHCG 9,
the break points of the derivative 2 seemed to be identical
and was located within the region near its centromere between
2q11.1 or 11.2 and 2p11.1 or 11.2 (Figures 10 and 11). The

exact position is difficult to determine by G-11 staining

36

alone because the centromeres of the human chromosome 2 is not
usually stained pink. Therefore both centromeric regions of
the hamster chromosomes and the human derivative 2 display a
pale blue color. If this specific rearranged chromosome
carried human derived centromere, the breakpoint would be then
assigned at 2p11.1 or 11.2: but if the centromere is Chinese
hamster oriented, the derivative 2 would be at 2q11.1 or 11.2.

The breakpoint in MHCG 11 was also located on the long
arm of the derivative 2, but at 2q23 (Figure 14).

In cell line MHCG 10, three kinds of karyotype were
found. Seven cells had the intact derivative 2 (Figure 15).
Four cells contained a part of the long arm of the derivative
2 which was broken at 2q21 or 22 and translocated to a Chinese
hamster chromosome (Figure 13) . The third type (including four
cells) involved a translocation between a Chinese hamster
chromosome and the distal part of the long arm of the human X.
The breakpoint was determined at Xq22 (Figure 12). The origin
of this rearranged Xq could be either from the normal X
chromosome or from the derivative 2. It is not possible to
distinguish one from the other by karyotypic analysis alone,
but the identification will become possible when the
restriction fragment length polymorphism technique is
employed.

4- WW9:

Nine out of 54 cells were found to carry neither the

37
human derivative 2 nor the derivative or normal X chromosome.
These included 4 cells in MHCG 1A, 1 cell in MHCG 9 and 4
cells in MHCG 10. In addition, four more cells in MHCG 10
retained the translocated Xq distal to the Menkes locus with
the breakpoint at Xq22.2. Therefore, the Menkes gene was

apparently missing from 13 out of 54 cells analyzed.

5. I 1f=‘ lau‘ - -Ou-sou‘ 1-n:‘ s 1 --. e V ,es:
Chinese hamster has 22 (11 pairs) chromosomes, of
which 10 pairs are autosomes and 1 pair is the sex chromosome.
In our four hybrid clones, 35-45 chromosomes were found per
cell from hamster origin in MHCG 1A. Most cells (19/22) had
near tetraploid chromosomes varying from 39 to 44. They were
probably the result of either fusion of two hamster cells with
a single human cell (Grzeschik et al, 19732") or the failure
of mitosis in the hybrid cells at early stage followed by
random loss of an individual chromosome from the same cell
later. In contrast, the numbers of hamster-derived chromosomes
were close to normal (22) in the other three clones: for
example, 20-23 hamster chromosomes were found in every cell in
MHCG 9. The origin of the extra chromosome in the cell with
the total number of 23 could possibly come from a breakage of
a hamster chromosome into two, therefore, the total number of
chromosomes increased to 23 instead of the normal value of 22.
In fact such a breakage was observed in MHCG 9 as one segment

of the hamster chromosome was rearranged with the human

38

Table 6 Numbers of Hamster Chromosomes in Each Cell Line

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

——
# of hamster # of
cell lines total
chromosome/cell cells
35 1
38 1
39 3
4O 3
MHCG 1A 22
41 5
43 3
44 5
45 1
20 1
MHCG 9 22 2 4
23 l
15 1
19 2
20 1
Ii
MHCG 10 21 9 19
22 4
43 1
45 1
20 2
21 1
MHCG 11 9
22 5
38 1

 

 

 

 

39

' derivative 2 to form a new chromosome. The similar phenomenon
was observed in MHCG 10, in which the majority of cells
(14/16) retained 19-22 hamster-derived chromosomes except one
which had 45 from the hamster. This specific spread might
represent that either two separate sets of chromosomal
contents from two metaphases overlapped with each other during
the process of making slide or a non-disjunction occurred at
a certain stage during' cell division. Two intact. human
derivatives 2 were found in this mitosis: In MHCG 11, most
spreads (8/9) reserved 20-22 hamster chromosomes. The
remaining cell was found to have 38 hamster chromosomes which

might be explained by the same reason as discussed above.

DISCUSSION

An X-linked recessive disorder affects males while
females are unexpressed carriers of the gene. Therefore, it
was unusual that full expression of the Menkes Syndrome was
observed in the female patient, C.G. , carrying an X-autosomal
translocation. It is likely that the normal X chromosome was
non-randomly inactivated in this patient while the rearranged
X remained active and the gene for Menkes was interrupted by
the translocation. As a result, expression of the Menkes gene
in this patient was defective. Support for this hypothesis
comes from comparative mapping studies from an animal model of
Menkes disease and linkage data in humans. The Mo locus
(‘mottled' in mouse) is equivalent to the Menkes gene in human
and was mapped to X chromosome between loci for ng-l and Ags,
and closely linked to ng-l. The gene order for these loci
with respect to the centromere was already known to be highly
conserved between mice and humans. The human PGK locus has
been mapped to band qu3 (Horn et a1, 1984). This band was
where the translocation breakpoint was located in the patient,
C.G.. Based on these findings, it is hypothesized that full
expression of Menkes disorder in this patient was due to
disruption of normal functions of the gene by the

translocation. The gene for Menkes was, therefore, most likely

40

41

located at band qu3. Recently, this case has been utilized by
investigators at the University of Michigan to precisely map
the Menkes gene in humans. Molecular DNA analyses using a
large number of probes from the human X chromosome confirmed
that the breakpoint in this patient was located in the region
Xq13.2-13.3, proximal to the PGKl locus (Verga et al“, 1990) .
However, another possibility exists that the locus for Menkes
disease might be somewhere on the rearranged X chromosome and
due to non-random inactivation of the normal X, this defective
gene was expressed. Under this hypothesis, the translocation
and dysfunction of the gene by a separate mutation are two
independent events that occurred in father's germline since
the rearranged chromosome was identified as paternal origin.
However, the possibility for two events arising independently
on the same chromosome is extremely low.

IX Somatic cell hybridization has provided a powerful
experimental approach to map the human genome. By growing
hybrid cells in a selective HAS media, we are able to isolate
clones retaining the human X chromosome(s) on which the
selective marker HPRT gene resides. Since one X chromosome is
usually randomly inactivated in females and, therefore, the
HPRT gene, the opportunity will be increased for the random
loss of the inactivated X chromosome with retention of the
active one, provided cultures are kept in the selective medium
for a prolonged period. Whether a gene of interest on X

chromosome is able to be retained in cells depends on the

42
distance between this gene and the selective marker and also
the length of the time which the clones are allowed tO>grow in
the selective medium. Previous studies have shown that
chromosome breakage was a frequent outcome when somatic hybrid
cells were kept in HAT selective medium for an extended period
of time (Siniscalco‘7, 1974), and it occurred more often
between two gene loci which were far apart than two loci which
were close to each other. Such a breakage would result in
mitotic separation of the gene of interest from HPRT locus on
the same X chromosome. Subsequently intra- or inter-species
chromosome rearrangements *will result in the loss of‘ a
particular gene despite the retention of the selective marker
HPRT in the clone. HPRT has been assigned at Xq26 and.the gene
for Menkes is assumed to be located at qu3. In the case of
the patient C.G., the long arm of the X distal to the
breakpoint qu3 was rearranged with chromosome 2 to form the
derivative 2. Therefore, the der 2 contained HPRT gene and
presumably a part of or the whole gene for Menkes. Based on
cytogenetic analysis, the der 2 was present in cells in two
forms: the intact and the translocated chromosome derived from
the broken der 2 containing loci for both qu3 and Xq26.
Either one of the two forms of the der 2, but not both, was
found in each clone of MHCG 1A, 9 and 11, which probably
indicated that the breakages and rearrangements of the der 2
occurred independently at an early stage of cell fusion in

each of those cell lines. However, it was observed that in

43

addition to the intact and translocated der 2, a few cells in
MHCG 10 did carry the distal part of long arm of a broken
chromosome X with the breakpoint at Xq22 .2 between the
presumed gene loci for Menkes and HPRT. This finding suggested
retention of the selective marker HPRT and loss of Menkes gene
from these cells. It is assumed that this piece of broken X
should bear the activated HPRT gene for HAT selection because
it was the only copy of X found in these cells. X chromosome
inactivation is known to be quite stable in humans and there
is little possibility for an inactive gene to become
reactivated (reviewed by Gartler et a1“, 1990). This means
that the inactivated normal X in this patient was unlikely to
be reactivated. It is reasonable to postulate, therefore, that
the distal Xq carried in MHCG 10 was likely from the der 2
broken at Xq22.2. Since a total of 8 out of 19 cells in MHCG
10 were found missing the Menkes gene, this cell line would
not be useful for the purpose of mapping the locus for Menkes
gene. The absence of the normal X in each cell line indicates
that it has been preferentially lost from the cultures due to
non-random inactivation of the entire chromosome including
HPRT gene.

Identification of human chromosomes against a mouse
chromosomal background by various staining methods is a prime
requirement for genetic analysis via somatic cell
hybridization. Use of G-11 staining technique is a reliable,

reproducible, fast and easy way to achieve this goal. Giemsa

44

banding followed by G-11 staining allowed us to compare
different banding patterns on the same chromosome so that the
origin of an individual chromosome can be distinguished. It is
especially useful in characterization of a chromosomal
translocation involving two different species. Under this
condition, the two-colored chromosome will be observed in a
well-stained metaphase, indicating the rejoining of the
differentspecie-specific DNA materials into one chromosome.

The mechanism of G-ll banding is not clear. Two
components of Giemsa are required to obtain the differential
staining. One is azure A or B, the other is eosin Y. It was
observed that. G—11 banding ‘was. highly' dependent on. the
concentration and ratio of two dyes, the pH of the stain
solution and the buffer composition (Wyandt et al, 1976).
Since the banding was achieved only when methylene blue and
eosin were used in the combined solution, it indicated that
the formation of a azure-eosinate complex accounted for the
striking magenta color of chromosome bands (Sumner et ar“,
1973). The role of alkalinity in stain solution is not clear.
It can only be speculated that the raised pH of the stain will
result in an increased amount of human chromatin losing the
capacity of forming magenta dye complexes (Borrow‘9, 1974) .' The
phenomenon to support this theory is that both mouse and human
chromosomes in hybrid cells are stained bright magenta at low
pH, but turn uniformly blue at high pH. Clearly the formation

a of red color is not because of a particular property of

45

constitutive heterochromatin or of highly repetitious DNA
sequences since mouse heterochromatin is stained the same
color as human euchromatin. The color difference on species-
specific chromosomes seems likely due to an alteration in the
structure of particular regions of chromatin by the effect of
pH on associated structural proteins rather than the direct
influence of pH on the process of dye binding (Borrow, 1974).
However, the real mechanism for the differential staining is
not well understood.

G-ll banding is a relative sensitive technique. Many
influences can be attributed to the staining quality, for
example: the type and lot number of Giemsa stain, the banding
method used, the age of slides and the manner they are aged,
the densities of cells on the slide, the type and pH of
different buffers, the length of time of staining and of pre-
soaking slides in alkali before the staining, etc. Erratic
staining results were observed by using, not only different
brands of Giemsa but also different bottles of the same brand
and lot number of the stain solution (Wyandtflfi 1976). The pH
of the buffer has the most notable influence on staining
quality of a slide and its optimum varied between different
cell types and different slides. Generally speaking, newly-
made slides need higher pH than aged slides to achieve maximum
differentiation: room temperature aged slides require longer
staining time than slides aged in 60'C oven: in order to

obtain an optimal banding pattern, slides made from different

46
clones may also need slightly different pH. The G-11 banding
procedure used in this research is modified from Wyandt's
method (1976). The Giemsa stains that Wyandt suggested were
Harleco, azure blend, type 42476 and MCB type GX85 3527. With
these blood stains, the best G-ll banding patterns on the
human chromosomes were observed at pH 11.3-11.6 and the
staining time around 10 minutes on the slides aged from 3 days
to one month. However, the optimal color differentiation on
hybrids MHCG 1A, 9, 10 and 11 in this experiment was not
obtained until pH of CAPs buffer raised to 11.98-12.03. This
difference may be explained by several reasons. The first is
that the Giemsa powder used to stain MHCG cell lines was
Fisher G-146, which was different from the Giemsa powder that
Wyandt utilized. This difference relied on the composition and
ratio of several common components, which often influence G-ll
staining quality. Secondly, G-11 banding was thought to be
involved in denaturation of a specific satellite DNA known to
be localized to magenta stained regions (Jones", 1973) .
Different species have different satellite DNA composition,
therefore a different pH was needed to achieve the structural

alteration required for color differentiation.

SUMMARY

Utilization of human strains with an reciprocal
translocation has become a valuable tool for mapping the human
genome via somatic cell hybridization. G-11 staining technique
has provided a useful approach to characterize the human
chromosome contents in human-mouse hybrid clones. Under an
appropriate pH of stain solution, human DNA materials can be
distinguished by its light blue color against dark magenta-
stained mouse or Chinese hamster chromosome background. Four
cell hybrids were tested by G-ll sequential staining technique
in this study to identify the translocated human chromosome Xq
for further mapping Menkes gene in humans. The majority cells
from MHCG 1A and 9 and all the cells from MHCG 11 were found
carrying this piece of X chromosome including the speculated
locus for Menkes (qu3). They were, therefore, usable for the
further gene mapping. In contrast, MHCG 10 is not an
appropriate cell line because the locus qu3 was found to be
missing from 42% of the cells in this cell line. Neither the
derivative nor the normal X was observed in all four clones on
the basis of cytogenetic analysis.

G-ll staining technique is relatively inconsistent.
Modification may be needed on the pH of the buffer and the

length of staining time for different slides and different

47

48
cell lines. It is strongly recommended that a minimum number
of slides made from each clone and cells photographed from
each slide is required to guarantee enough well-stained

metaphases for further analysis.

49

r

I,“
sfifinfiffi e...

.5 I\
null:

0. V.
1.. I.
.0.‘ 1‘...

 

it o
e . an
he r
90...!” .IO 051.
o_ . . v
.nv’ .\ be . § ’11.

a:

.
~M4.-w.n~a§0 M #5”!

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0 M
We ‘00 .
a ‘u
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ahw‘ .

(Arrows indicates the breakpoints of the chromosomes

Figure 1 Blood karyotype of the patient C.G.
2 and X)

 

 

 

50

 

Figure 2 Chromosomes stained by G-ll technique
(Arrow 1 indicates an entire human chromosome. Arrow 2
marks a translocated chromosome. The part to the left
of the arrow is of human origin and the part to the
right of the arrow is of mouse origin)

.‘jl

 

Figure 3a G-ll stained metaphase showing human chromosome
composition in MHCG 1A
(Arrow 1 marks the rearranged mouse/human derivative 2 .
The portion to the right of the arrow represents part
of the translocated human der 2. Arrows 2 indicate
other human chromosomes)

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

52

Figure 3b Giemsa-banded metaphase Showing human chromosome
composition in MHCG 1A
(Arrow 1 indicates the rearranged mouse/human
Derivative 2: Arrows 2 show the human chromosomes 14.
Arrows 3 mark the human chromosomes 22)

 

 

 

 

 

 

 

 

Figure

4a G-11 stained metaphase showing human chromosome
composition in MHCG 9

(Arrow 1 indicates the rearranged mouse/human
Der1vative 2. The portion to the right of the arrow is
part of the derivative 2. Arrow 2 indicates the human
chromosome 14)

54

Figure 4b Giemsa-banded metaphase showing human chromosome
composition in MHCG 9
(Arrow 1 marks the translocated mouse/human chromosome.
The derivative 2 is the part to the right of the arrow.
Arrow 2 indicates the human chromosome 14)

 

 

 

 

55

Figure 5a G-ll stained metaphase showing the intact human
derivative 2 present in MHCG 10
(Arrow indicates the human derivative 2)

Figure 5b Giemsa-banded metaphase showing the intact human
der 2 present in MHCG 10
(Arrow indicates the human derivative 2)

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

57

Figure 6a G-11 stained metaphase without the derivative 2
from MHCG 10 (Arrow indicates the human chromosome 8)

Figure 6b Giemsa-Banded Metaphase without the derivative 2
from MHCG 10 (Arrow indicates the human chromosome 8)

 

 

 

 

59

Figure 7a G-11 stained metaphase showing the translocated der
2 in MHCG 10
(Arrow 1 indicates the translocated mouse/human
derivative 2. The light portion to the left of the
arrow is of human origin. The dark portion to the right

of the arrow is of mouse origin. Arrow 2 marks the
human chromosome 8)

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

60

 

Figure 7b Giemsa banded metaphase showing the translocated
der 2 in MHCG 10
(Arrow 1 indicates the translocated mouse/human der 2.
The portion to the left of the arrow is of human
origin. The portion to the right of the arrow is
from mouse. Arrow 2 indicates the human chromosome 8)

 

 

 

 

 

 

 

61

,.4’
C

v
-J

Figure 8a G-11 stained metaphase showing the translocated Xq
in MHCG 10
(Arrow 1 indicates the translocated mouse/human X. The
light portion above the arrow is of human origin and
the dark portion below the arrow is of mouse origin.
Arrow 2 marks the human chromosome 8)

 

 

 

 

 

 

 

 

 

 

 

62

Figure 8b Giemsa banded metaphase carrying the translocated
Xq in MHCG 10
(Arrow 1 marks the rearranged mouse chromosome/human X.
The part above the arrow is from the human chromosome

X. The remaining part is of mouse origin. Arrow 2
indicates the human chromosome 8)

 

63

 

Figure 9a G-ll stained metaphase showing human chromosome
composition in MHCG 11
(Arrow marks the translocated mouse/human derivative 2 .
The chromosome portion to the right of the arrow is of
human origin and the portion to the left of the arrow
is of mouse origin)

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

64

Figure 9b Giemsa banded metaphase showing human chromosome
composition in MHCG ll
(Arrow marks the rearranged mouse/human derivative 2.
The part of the chromosome to the right of the arrow is
of human origin and the portion to the left of the
arrow is of mouse origin)

 

65
E
i\\
rii" g o s -
'123535:==
der 2 t(der 2) 14 14 22 22

 

f

{Elztrx

/:,\ . "

'i . ib. ' 0' I;
i

.

«f “x \
:1")

iiililiii""”'

x:
0.
we».
03-3.

Figure 10 Karyotype of MHCG 1A
(The translocated derivative 2 and the human
chromosomes 14 and 22 are shown at the top of the
karyotype. The mouse chromosomes are at the bottom and
arranged in order of the length. Arrow marks the
junction point between the mouse and human chromosomes)

66

 

 

 

 

 

 

 

rn- F:
.‘j: = .
. - .’
II. —
33‘-
.?i
l‘ -g @
Zu=t:; 7::
" ‘i‘ : b
’ ”.
El .
t. ‘ i w
t”: >”
.E' ___—————-—"
der 2 t(der 2) 14

 

 

 

Figure 11 Karyotype of MHCG 9 showing the translocated
derivative 2
(The chromosomes below the double-line are of mouse-
origin. The human chromosomes are above the double-
line. Arrow indicates the junction point between the
mouse and the human derivative 2. The part below the
arrow is of human origin and the part above the
arrow is from the mouse)

 

 

 

 

 

 

 

 

 

 

 

 

Figure 12 Xaryotype of MHCG 10 showing the rearranged human
Xq
(The human chromosomes are at the top of the karyotype.
Arrow indicates the junction point between the mouse
and human chromosome. The chromosome portion below the
arrow is from the human X)

68

A“‘_
.. >1 l
a

lrmnhlnmlmnnsll

der 2 t(der 2) 8

 

 

V.;.!Ii"“9l. IV
s“
~‘IdL,AI‘
~b 1"“

:m sun-2'

Figure 13 Karyotype of MHCG 10 carrying the translocated
human derivative 2
(The derivative 2 is showed as the portion of the
chromosome below the arrow. The mouse chromosomes are

under the double-line)

 

69

until ss’x‘millsuf

f)

‘0' uN-fil ' o

 

der 2 t(der 2)

 

Figure 14 Karyotype of MHCG 11 showing the translocated
derivative 2
(The human chromosome is at the top of the karyotype.
The portion below the arrow is part of the human der 2.
The mouse chromosomes are under the double-line)

70

 

 

 

Figure 15 Karyotype of MHCG 10 carrying the intact derivative

2
(The human chromosome is above the double-line and the

mouse‘chromosomes are below the line)

 

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11.

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