#4 i I \ 1- n: $.11»- , _‘_‘ u.. «vaov .-~n¢ -,... -‘_ 'h’ - . """5‘ :n'¢‘l"~ « 45“”1: u» . .~ ~ ~¢f€”:x~7‘c?—r§z‘ ‘ .t V A“ . A 21.. “ESQ-K w‘ ""‘1'uie "4'5. 1 ' 03‘3ww4. 'H _. .5... - . . r ”7‘ . r - x- "c --r 5,7253%}; .4".- r";‘1‘ .,. ,,..: .- ~r~ 9‘ 'Jc...,..r..;:.~u;_ "a: J‘. .2341... ' -1‘.’ 5L .4 3...“..«4 . ma-I'VI! :- . u... .. , ,N. ...... .. ....- I-» . . ..:. -..:...,‘.. ""‘ ' 3:757:3'“ w. » v , $2....«..;,.-<..~ ”a“. 9...- _ V...-...-:--4“-_ ' ' "(,7 (’- .—-—"'S- w- {r W” w'-“-::r"..,..'.r.:..:r. "‘4‘- .. :‘f-w'“ ‘ . ”gun-‘0' noupvomw~ -m. n—Awa- '3 MM”-.. W’s: H 1..-. ,~ a” LWN; ’4. —-p' ma ~-.- A- .— v- .,_..n-- v _, A..- u,» I: , auawm , ~-——"““"":..._:w ‘ . wua-w 4"“ . (”v-,4. vflvrv" - "" " Mom» I 3,123:- ..o. “0- ' 1" #7 £th “- -.. “M Wu" :5- ‘ "‘ wa. ‘ it ._ -. . “wag 7- ’ r on”,— Mr- M... a. ,. ~.-,, .. ”an"- .. n... .1“... n .m- rum.-. 0. H...” "r l *4- 1%-,- - .n. A. wan—w . ..... 4vu1wrr.‘ ... .«M M a.“ . ... ,. w. ... v..- dlv‘rm I’- M .a m..«w-...$.‘4 "7"... ' "t‘.."'.§'.’,‘."v7::' ..-,.. -V», o...— , hLu—x "Mn-M , ”no- rug—~- nagw up" -- - ---..........,4..m... .. ”ca "m. N ”Or—'9‘.“ ya...“ _ WW" 'r Idfl; u . .1.“ a v. llllmllllllllllHIHHHIHIIHIHllllllllllllHlllllllllll _ 300784 1103 infiv " LIBMRY Riemann State University k . This is to certify that the thesis entitled Differential Expression of ras Genes During Growth and Development in Mucor racemosus presented by Sung-Yuan Wang has been accepted towards fulfillment of the requirements for M' 5' degree in We 04m 5’ Dr. John E. Linz Major professor Date //‘8”8 3,500 with several changes of TE buffer (10 mM Tris-Cl and 1 mM EDTA, pH 8.0) for 24 hours. The concentration of the DNA solution was quantitated by spectrophotometric measurement. A260=1 unit corresponds approximately to 50 ug/ml for double-strand DNA. A pure preparation of DNA has an Azso/Azao ratio of 1.8. A single plaque of Bacteriophage M13mp18 was inoculated. into 10 ml of YT broth containing 5 ul of an overnight culture of JM101. At the same time, 100 pl of overnight culture of JM101 was inoculated into 100 ml of YT growth medium. Both cultures were incubated at.37”C with vigorous shaking for 3 hours. One ml of the phage—infected culture was then mixed with 100 ml of the JM101 culture to obtain a 25 multiplicity of infection of 1 to 10 and incubated for an additional 5 hours at 37°C with shaking. The cells were collected by centrifugation at 8,000xg (Sorvall SS-34 rotor) for 5 minutes at 4W3. Because the replicative form (RF) of bacteriophage M13 occurs in the bacterial cell as a circular double-strand DNA molecular, phage DNA was isolated, purified and quantitated by the same procedure as that for plasmid DNA. Subcloning DNA Fragments of Mugg; RAS Genes in Plasmid pUCi9 for use as Gene Specific Probes DNA restriction fragments containing the 3' hypervariable region of MRASl and MRAS3 genes were subjected to restriction endonuclease digestion of plasmids containing the MRAS genes. The restriction fragments were resolved by electrophoresis on a 1% agarose gel and purified by electroelution using an apparatus from International Biotechnology Instrument (IBI). Ligation was carried out as described by Maniatis et al. (1982). Vector DNAs (plasmid pUC19) were linearized by incubating the plasmid with restriction endonucleases which would generate ends complementary to the cloned MRAS DNA fragment. A 3:1 molar ratio of MRAS DNA fragment to 100 ng of linearized plasmid pUC19 DNAs were mixed with 2 pl of 10x ligation buffer (0.5 26 M Tris pH 7.5, 0.1 M MgClz, 0.1 M dithiothreitol, 10 mM spermidine, 10mM ATP, 1 mg/ml DNA enzyme grade bovine serum albumin) and 10 units of T4 DNA ligase. The mixture was made up to a final reaction volume of 20 pl with water. Two microliters of the preincubation mixture was saved for ligation analysis by 1% of agarose minigel. The reaction mixture was incubated at 16°C for 12-16 hours. After ligation, 2 pl of the reaction was removed to analyze for successful ligation by gel electrophoresis. The remaining ligation mixture was then diluted with TE buffer (10 mM Tris-Cl, 1 mM EDTA; pH 8.0) to 80 pl and saved for transformations. Transformation of Plasmid and Bacteriophage into £1 9911 E; coil DH5a was the host strain for plasmid DNA. Competent cells for plasmid transformation were prepared by the Calcium Chloride procedure (Hanahan 1983, Maniatis et al. 1982). A single colony of E; ggli DHa grown on LB agar (Luria-Bertani, 1% wt./vol. Bacto-tryptone, 0.5% wt./vol. Bacto-yeast extract, 10 mM NaCl, pH 7.5 with 1.5% wt./vol. Bacto-agar) was inoculated into 3 ml of LB medium and incubated with shaking at 37°C overnight. The overnight culture was diluted 1/200 in LB medium and incubated with shaking at 37°C until mid log phase (OD590=0.4-0.6. ca.2-3 27 hours). Cells were harvested by centrifugation at 7,000xg for 5 minutes at.43C and resuspended in 1/5 volume of ice cold 0.1 M MgClz and kept on ice for 15 minutes. The cells were then collected by centrifugation (7,000xg, 5 minutes at 43C) and resuspended in 1/50 original volume of ice cold 0.1 M CaCl2 and chilled in an ice bath for 60 minutes to generate competent cells. Samples of diluted (4-fold dilution) ligation mixture (5 pl and 20 pl) were added to separate tubes containing 100 pl of competent cells, and mixed by gently tapping the tubes. The transformation reaction was kept in an ice bath for 30 minutes allowing the cells to take up DNA. The transformed cells were heat shocked for 2 min at 42°C and then placed on ice for 2 min. Transformed cells were recovered by adding 1 ml of SOC (2% wt./vol. Bacto-tryptone, 0.5% wt./vol. Bacto-yeast extract, 10 mM NaCl, 25 mM KCl, 20 mM MgSOJMgClz (2 M Mg” filter sterilized stock), 20 mM Glucose (2 M filter sterilized stock): mixed and filter steriled) at rm. temp. and incubated with shaking at.37°C for 60 minutes. Aliquot (100 pl and 1 m1 concentrated to 100 pl) of the transformed cell suspension were spread onto LB agar medium containing ampicillin (50 pg/ml) and incubated at 37°C overnight. 3; ggli strain K12 JM101 was used as a recipient strain for transformation by M13mp18 and mp19 to propagate this bacteriophage. Cells were first grown on minimal agar 28 medium (500 ml of steriled 1.2% wt./vol. NazHPOU 0.6% wt./vol. KHZPO“ 0.1% wt./vol.NH,.Cl, pH 7.4 M9 salts combined with 500 ml of 3% autoclaved Bacto-agar: to this solution, 1 ml 1.0 M MgSO“ 1 ml 0.1 m CaClz, 1 ml 1.0 M Thiamine-HCl and 5 ml 40% glucose were added) to ensure that the F episome of the bacteria was not lost. Competent cells of E. ggli K12 JM101 were generated by a procedure similar to that for competent cells of g. 9911 DHSa. JM101 cells were grown on YT medium (0.8% wt./vol. Bacto-tryptone, 0.5% wt/vol. Bacto-yeast extract, 0.5% wt./vol NaCl and adjusted to pH 7.4, 1.5% wt./vol. Bacto-agar was added for agar plates before autoclaving). An aliquot (3 pl) of bacteriophage DNA was added to 100 pl of competent cells and incubated on ice for 40 minutes with occasional shaking. The cells were then heat shocked at 42°C for 2 minutes. One pl, 10 pl and 90 pl of the transformation reaction were added to 3 tubes containing 0.2 m1 of plating culture which was generated by inoculating a single colony of JM101 into 3 ml of YT broth and incubating at 37°C until late-log phase of growth (OD550=1.0) . Molten YT medium (3 m1, 45°C) containing 0.1 ml 2% (wt.vol.) X-gal (5-bromo-4-chloro-3-indolylbeta-D- galactoside in dimethylformamide) and 0.2 ml 100 mM IPTG (isopropyl-beta-D-thiogalactopyranoside) was added and quickly mixed in each tube. The mixtures were then poured onto YT agar medium. The plates were kept at room temperature for 15 minutes to allow the top agar to harden 29 and then incubated at 37°C overnight. Radiolabeling DNA Restriction fragments DNA restriction fragments to be used as gene-specific DNA probes for hybridization were gel purified by electrophoresis on agarose gels and electroeluted from gel slices. DNA fragments were denatured by heating to 95-100%: in a sealed eppendorf tube for 5 minutes and immediately chilled to 0°C in an ice bath for 5 minutes. The random primer labeling procedure (Feinberg and Vogelstein 1983) was used to radiolabel the probes. The reaction was carried out by mixing H53 (to a total volume of 50 ul) , 13 ul of oligo- labeling buffer (OLB: 10 pl mixture of 1.8% 2- mercaptoethanol :2 M Hepes:1 ug/ml hexadeoxyribonucleotides (2:5:3) and 3 pl of 1 mM dATP, dCTP and dTTP dissolved in TE buffer pH 7.0, 10 mM MgCl2 mixture), 2 pl of 1 mg/ml solution of bovine serum albumin (molecular biology grade, BRL), 10-30 ng of denatured DNA, 5 pl of [aQRP1dGTP (DUPONT/NEN research products, 3000Ci/mmol, 10 mCi/ml) and 2 units of large fragment of E; 2211 DNA polymerase I (Boehringer Mannheim Biochemicals; labeling grade Klenow fragment). The complete reaction was incubated at rm. temp. at least 2 hours. The reaction was stopped by adding 1 pl of 0.5 M EDTA. The labeled probes were purified by gel 30 exclusion column chromatography (Sephadex G50-80, 5 ml of packed volume) by elution with TE buffer (pH 8.0). The radiolabelled DNA was collected in the first peak detected with a Geiger counter. Two aliquots (5 ul) of eluted DNA were spotted onto glass-fiber filters. The DNA sample on one filter was precipitated with Trichloroacetic acid (TCA) (Maniatis et al. 1982). The specific activity of the labeled DNA was then quantitated by liquid scintillation spectroscopy (United Technologies, MINAXI TRI-CARB 4000 series scintillation counter). A high specific activity (for example 1 x 108 cpm/pg DNA) was achieved by using as little as 10 ng of DNA in each reaction. Selection and Screening for the Presence of Recombinant Plasmids in 59 c011 cells The desired recombinant from a population of bacteria was isolated by genetic selection and in situ hybridization of bacterial colonies. The plasmid pUC19 carries an ampicillin resistance gene and a multiple cloning site within the fi-galactosidase gene. It is therefore possible to screen E9 9911 cells for the presence of a recombinant plasmid by selecting for resistance to the antibiotic ampicillin and insertional inactivation of the fi- galactosidase gene. Bacteria which carry an active B- 31 galactosidase gene on plasmid pUC19 can hydrolyze the chromogenic substrate, X-gal, and generate a blue colony. A DNA fragment inserted into the B-galactosidase gene of pUC19 inactivates the gene. Cells containing these recombinant plasmids appear white on medium supplemented with X-gal. For in situ colony hybridization (Grunstein and Hogness 1975, Maniatis et al. 1982.), randomly selected bacterial colonies suspected to carry recombinant plasmids were spotted by a toothpick onto a nitrocellulose (N.C) filter (Schleicher & Schuell) which was placed on LB/ampicillin agar medium and incubated at.37”C overnight. The filter with bacterial colonies was placed with the colony side up on a sheet of 3MM filter paper saturated with 10% SDS (sodium dodecyl sulfate) solution for 5 minutes to limit the size of hybridization signal and then transferred onto another sheet of 3MM filter paper saturated with base solution (1.5 M NaCl, 0.5 M NaOH) for 5 minutes to denature the DNA. The filter was transferred to a third sheet of 3MM filter paper saturated with neutralizing solution (1 M Tris- Cl pH 8.0, 1.5 M NaCl) for 10 minutes and then transferred onto a 3MM filter paper saturated with 2x SSC solution for 10 minutes.. The filter was air dried for 60 minutes and baked at 80°C for 2 hours under vacuum. The DNA on the filter was hybridized with a gene-specific probe to detect recombinant clones. 32 Purification of mRNA from g. :999g9999 RNA from £999; cells of several morphologies was prepared by the procedure of Maramatsu (1973). The glasswares used for RNA purification were treated with 0.1% of diethylpyrocarbonate (DEPC) and baked at 250°C for 12 hours. Cells were placed into a sterile mortar containing liquid nitrogen and broken by grinding with a pestle for 10 minutes. The frozen cell material was transferred to an ice cold 30 ml Corex tube and resuspended with 1 ml of cold SDS- RNA-extraction buffer (50 mM sodium acetate, 1.0 mM EDTA and 1% SDS: adjusted to pH 5.0 with glacial acetic acid and treated with DEPC). The suspension was then mixed with an additional 4 ml of SDS-RNA-extraction buffer warmed to 65%: followed immediately by 5 ml of hot phenol (65°C saturated with buffer, containing 0.1% wt./vol. 8-hydroxyxyquinoline and 0.2% fi-mercaptoethanol). The samples were vortexed for 30 seconds. The mixture was placed at 65°C for 15 minutes. The aqueous phase containing the RNA was recovered and extracted with 1 volume of phenol:chloroform:isoamyl alcohol (25:24:1) and then with 1 volume of diethyl ether (water saturated). The aqueous phase was then mixed with 1/6 volume of 3 M sodium acetate followed by an additional 2.5 volumes of ethanol. The RNA was allowed to precipitate by storing the solution at -20°C overnight. It was recovered by centrifugation at 5,000xg for 10 minutes at.4PC. The 33 pellet was washed with 75% ethanol containing 0.1 M sodium acetate (pH 7.5). The purified total RNA was dissolved in TE buffer (pH 8.0). The concentration and purity of RNA were measured by spectrophotometric determination. An Ann of 1 corresponds to approximately 40 ug/ml of RNA. A pure preparation of RNA has an Azso/Azso ratio of 2.0. Polyadenylated RNA was separated from nonpolyadenylated RNAs by oligo(dT)-cellulose chromatography (Edmonds et al. 1971, Aviv and Leder 1972, Maniatis et al. 1982). Dry powder oligo(dT)-cellulose (0.25 g) was used to pack a 1 ml column for 10 mg of total RNA. The column was prepared by equilibrating the oligo(dT)-cellulose powder in sterile loading buffer (20 mM Tris-Cl, pH 7.6: 0.5 M NaCl, 1 mM EDTA and 0.1% SDS). The solution was then poured into a sterile silanized pasteur pipette plugged with silanized glass wool. The column was packed by washing with 3 column-volumes each of sterile water, 0.1 M NaOH and 5 mM EDTA solution, and sterile water (until the pH of the column effluent was less than 8) and then with 5 volumes of sterile loading buffer. The RNA sample was dissolved in sterile water and heated to 65°C for 5 minutes following the addition of an equal amount of 2x loading buffer. The sample was cooled to rm. temp. and loaded onto the column. The effluent was collected, heated to 65°C, cooled and reapplied to the column. The column was then washed with 5-10 column-volumes of loading 34 buffer followed by 4 column-volumes of loading buffer containing 0.1 M NaCl. The poly(A)'RNA was eluted with 2-3 column-volumes of sterile elution buffer (10 mM Tris-Cl, pH 7.5: 1 mM EDTA and 0.05% SDS). Samples containing poly(A)‘RNA were pooled and sodium acetate (3 M, pH 5.2) was added to a final concentration of 0.3 M. The RNA was allowed to precipitate with the addition of 2.2 volumes of ethanol and stored at -20°C overnight. It was recovered by centrifugation (5,000xg,4°C for 10 min) and dissolved in sterile water (DEPC-treated). Restriction Bndonuclease Analysis and Gel Electrophoresis Restriction endonuclease digestions of DNA were carried out according to the optimal reaction conditions as described by manufacturers. DNA restriction fragments were separated by gel electrophoresis. Gels varied in percentage of agarose (0.8%-1.0%) depending on the size of DNA fragments under analysis. A Tris-acetate buffer system (TAE: 0.04 M Tris-acetate, 0.02 M EDTA) (Maniatis et al. 1982) containing 0.5 pg/ml of the fluorescent dye ethidium bromide (Sharp et al. 1973) was used to make up agarose gels and for the electrophoresis buffer to stain the separated DNA fragments. Alternatively, 0.5 pg/ml ethidium bromide solution was applied upon completion of electrophoresis in a 35 45 min soak. DNA fragments separated in the gel were visualized with a short wave (254 nm) UV-light transilluminator and photographed through a yellow filter with Polaroid type 667 film. RNA samples were fully denatured by treating the sample with formaldehyde and formamide and resolved by electrophoretic separation through 1% to 1.5% formaldehyde- agarose gels (Maniatis et al. 1982, Fourney et al. 1988) with a MOPS/EDTA buffer system (10x stock solution: 0.2 M MOPS (3-(N-morpholinolino)-propanesulfonic acid), 50 mM sodium acetate, 10 mM EDTA, pH 7.0: treated with 0.1% DEPC and autoclaved). After electrophoresis, one lane on the agarose gel containing a commercial size marker RNA ladder (Bethesda Research Labs) was separated from the other RNA samples and post-stained with 0.5 pg/ml ethidium bromide in 10 mM MgSO, solution for 30 minutes. The RNA ladder was photographed on a short wave UV-light (254 nm) transilluminator through a yellow filter with Polaroid type 667 film. The formaldehyde in the sample gel was removed by soaking the gel in 1 mM MgSO, solution for 60 minutes. 36 Southern and Northern Analyses of Nucleic Acid Similarities between particular sequences of DNA were analyzed by Southern transfer and hybridization (Southern 1975, Southern 1980, Maniatis et al. 1982). DNA fragments were separated by electrophoresis through agarose gels and denatured by soaking the gel in several volumes of base solution at room temperature for 60 minutes. The gel was then neutralized with 3 changes of the neutralization solution with constant shaking at room temperature for 60 minutes. Denatured DNA fragments which were greater than 300 base pairs in size were generally transferred and immobilized to a nitrocellulose filter (Schleicher & Schuell). A Nylon membrane filter (Schleicher & Schuell, Nytran) was used to immobilize smaller DNA fragments. A 10x SSC buffer system was used for transfer of DNA by capillary action and allowed to proceed for 12 to 24 hours. The filter with bound DNA was air dried for 60 minutes. The DNA on the filter was then immobilized by baking the filter at 80°C under vacuum. Alternatively, DNA was bound on the nylon filter covalently by exposing the DNA on the filter to a short wave (254 nm) UV transilluminator for 5 minutes. The RNA was transferred to nitrocellulose filters or nylon membranes with 10x SSC by capillary action for 12 to 24 hours, and fixed to the membrane by baking for 2 hours at 37 80°C under vacuum or by placing nylon membranes with the RNA side face on a short wave (254 nm) transilluminator for 5 minutes. Southern and Northern blots for hybridization were soaked in prehybridization solution containing deionized formamide (40% for low stringency hybridization, 50% for high stringency), 5x Denhardt solution (5% ficoll, 5% bovine serum albumin, 5% polyvinyl-pyrrolidone), 6x SSC solution, 100 pg/ml denatured salmon testis carrier DNA and 5 mM EDTA ‘for 2 to 4 hours with shaking at rm. temp. For Southern analysis, 1-5 x 105 cpm/ml of a radiolabeled gene-specific DNA probe was then added to the prehybridization solution. For Northern analysis, 5 x 105 cpm/ml to 1 x 10° cpm/ml of radiolabeled gene-specific probe in hybridization fluid was utilized. The hybridization was carried out at 37°C (low stringency) or 42°C (high stringency) in a shaking water bath for 12 to 36 hours. Following hybridization, the filters were washed twice in 2x SSC/0.1% SDS washing solution at room temperature for 15 minutes followed by 0.1x SSC/0.1% SDS washing solution for 60 minutes at 42°C for a low stringency wash or 65°C for a high stringency wash. The filters were air dried and sealed with plastic wrap and exposed to Kodak XAR5 diagnostic film with or without an intensifier screen for 2 hours to several days depending on the intensity of hybridization signal. The image of the 38 hybridization signal on X-ray film was developed by soaking the film in Kodak developer for 5 minutes and a Kodak rapid fixer for 5 minutes. Image Analysis of Autoradiography The image of Southern and Northern hybridization signals on autoradiographs was analyzed by a video densitometer (Biomed Instruments, Inc.). The intensity of these hybridization signals was integrated as the scanned area of hybridization signal in a unit of pixel square. Dot Matrix analysis of Nucleotide sequences The similarity of nucleotide sequences was compared by a computer assisted dot matrix analysis. This method was develOped by Dr. George Gutman and Brian Ward, University of California, Irvine. RESULTS The research hypothesis of this study is that if one or more M999; 999999999 genes (MRAS) are involved in regulation of morphogenesis, the expression levels of the genes may correlate with changes in morphology. Therefore, it was necessary to specifically measure the expression of individual MRAS genes. The nucleotide sequences of 3 MRAS genes have been previously determined. Based on these data we were able to generate restriction maps of MRASl, MRASZ and MRAS3 and to predict the intron/exon structures of these genes (Figure 1). The nucleotide sequence data were compared to select regions among the 3 MRAS genes with a low degree of similarity. A sequence comparison was conducted using computer assisted dot matrix analysis and was based on a 8 bp match in a 10 bp window (80% stringency). The hypervariable region of the MRAS genes with less than 20% sequence similarity was identified in exon V located at the 3' end of each gene. Because of difficulty in identifying and purifying DNA restriction fragments containing only the hypervariable region (exon V) of each MRAS gene, longer restriction fragments which included part of exon V of each MRAS gene along with additional DNA sequences were selected to prepare DNA probes for analysis of MRAS transcript levels. 39 Figure 1. 40 Predicted intron/exon structure and restriction maps of Mgcor 999999999 MRASl, MRASZ and MRASB genes. Solid and hatched segments represent the predicted exons.Probe 1, probe 2 and probe 3 were used for detection of MRAS gene transcripts. Probe 1, 310 bp fing/E99RV restriction fragment (MRASl-l). Probe 2, 580 bp EanI/figmHI (figmHI site not shown) restriction fragment (MRASZ-O). Probe 3, 450 bp 999I/fl199III restriction fragment (MRASB-Zl). 41 HRA51 IV III II Haom >moom Han—m dam Haom Hflnm Haoz 1— Probe HRASZ IV III II HHHskd HHShd HHs>m Hanan 495m Honz >Moou Hsun HHUGHM HHHmk< Probe Z — NRAS3 IV III II HHHuafim Henm Hooz HHS>m Hudm Hadm PrObQ 3 — 42 Subcloning DNA Restriction Fragments of 5999; RAS Genes into Plasmid pUC19 MRASl. A 310 base pair KpnI/E9QRV restriction fragment containing a portion of the predicted hypervariable region of exon V and all of exon IV of the MRASl gene was subcloned into the plasmid vector pUC19 which was linearized with the complementary restriction endonucleases Kng/gmgI. The resulting recombinant plasmid was transformed into 39 9911 strain DH5a. The transformants were screened by the in situ colony hybridization technique using the 310 bp fing/E99RV DNA restriction fragment of MRASl gene (Figure 1) as a DNA probe. Eight of 72 bacterial colonies screened were found to have a recombinant plasmid containing the 310 KggI/E99RV restriction fragment of the MRASl gene (Figure 2). MRASZ. A 580 bp EygII/ggmHI restriction fragment containing the entire exon V of MRASZ gene and 3' 300 bp of flanking sequence was cloned into bacteriophage M13mp18. This subcloned restriction fragment had been used for analysis of the MRASZ nucleotide sequence (contributed by Dr. William Casale). MRA83. A 450 bp 9991/31991II restriction fragment, which contains the predicted exon III, IV and most of the exon V hypervariable region of MRAS3 (Figure 1) was also Figure 2. 43 In situ colony hybridization analysis of colonies transformed with MRASl-l. Bacterial colonies transformed with recombinant plasmids were screened by using the 310 bp KpnI/E99RV DNA restriction fragment of MRASl gene as gene- specific probe. Nitrocellulose filter was washed under low stringency conditions (0.1XSSC/0.l%SDS solution at 42°C for 60 minutes). The filter was then exposed to X-ray film without an intensifier for 4 hours at rm temp. Colonies marked + represent the positive control bacteria carrying recombinant plasmid containing the MRASl gene. Colonies marked - represent negative control bacteria carrying plasmid pUC19 only. 44 45 cloned into plasmid pUC19 which had been linearized by 999I/H1QQIII restriction endonucleases to generate complementary ends. The recombinant plasmid was transformed into E9 9911 DHSa. Transformants containing the recombinant plasmid were screened by colony hybridization using a 450 bp 999I/fi1991II fragment of MRAS3 gene (Figure 1) as a probe. Selected transformants (72 colonies) were screened and 8 clones showed positive hybridization (Figure 3). Analysis of Subcloned Fragments from MRASl-l, MRAsz-o and MRAS3-21 Recombinant plasmids containing the subcloned MRASl DNA fragment were prepared from several transformants by the alkaline lysis miniprep procedure and analyzed by restriction endonuclease digestion with KpnI/figmHI and E99RI/999HI. In the correct plasmid construct, these pairs of enzymes were predicted to generate 310 bp and 330 bp restriction fragments, respectively. These restriction endonuclease fragments were resolved by agarose gel electrophoresis (Figure 4). Only two transformant clones carried a recombinant plasmid containing a 310 bp Kng/figmHI fragment (figmHI site located in the polylinker region of the plasmid) or a 330 bp E99RI/fi9mHI fragment (both restriction sites located within the polylinker region of plasmid). One Figure 3. 46 In situ colony hybridization analysis of colonies transformed with MRAS3-21. B. 9911 DH5a cells were transformed with a recombinant plasmid containing a 450 bp SacI/fi199III restriction fragment of MRAS3 gene and screened by using a 450 bp 999I/fi1nglII DNA restriction fragment as DNA probe. Nitrocellulose filter was washed under low stringency conditions (0.1XSSC/0.1%SDS solution 42°C for 1 hour). The washed filter was then exposed to X-ray film for 4 hours without an intensifier at rm. temp. Colonies marked + represent transformants containing the MRAS3 gene as a positive control. Colonies marked - represent negative control bacteria carrying plasmid pUC19 only. 47 Figure 4. 48 Restriction endonuclease analysis of eight transformants carrying recombinant plasmids containing the subcloned MRASl DNA fragment. Plasmid constructs from transformants (1 through 8) were digested with restriction endonucleases gpgl and figmHI (lane a) or E99RI and figmHI (lane b), respectively, and the DNA fragments resolved on a 1% agarose gel. Lane x: Lambda DNA-HindIII digest size marker: lane y: PhiX174 RF DNA-flggIII digest size marker (New England BioLabs). 49 50 transformant was selected and large amounts of recombinant plasmid were prepared by cesium chloride density gradient centrifugation. The resulting recombinant plasmid was digested with gng and figmHI restriction endonucleases in combination to produce a 310 bp restriction fragment (Figure 6a). Southern analysis of this blot showed the presence of a 310 bp gpgl/ggmHI restriction fragment hybridized with a DNA probe prepared from MRASl KpnI/E99RV restriction fragment (figure 6a). This experiment demonstrated that the 310 bp £99I/999RV restriction fragment was subcloned in plasmid pUC19 and transformed into 91 coli DHSa. This recombinant plasmid containing the subcloned DNA fragment of MRASl gene was assigned the name MRASl-l. A 580 bp gygll/ggmHI DNA restriction fragment containing exon V and about 300 bp of flanking DNA from MRASZ (Figure 1) was previously subcloned into bacteriophage M13mp18 (W. Casale unpublished data). E1 9911 JM101 was infected with this recombinant phage to propagate the phage DNA. Restriction endonuclease analyses (E99RI and figmHI; 999RI site located in polylinker region of Mp13mp18) were used to confirm the structure of the recombinant phage (Figure 6b). The size of the restriction fragment was predicted to be about 600 bp (20 bp were added from polylinker region), but the resolved restriction fragment appeared slightly larger than 600 bp, possibly due to the Figure 5. 51 Restriction endonuclease analysis of eight transformants carrying recombinant plasmids containing the subcloned MRA83 fragment. Three different double restriction endonuclease digestions, §99RI and HindIII (lane a), EXQII and HindIII (lane b) or 2191 and E99RI (lane c) were conducted and the DNA fragments resolved on a 1% agarose gel to analyze recombinant plasmids prepared from 8 MRASB subclone transformants (1 through 8). Lane x: Lambda DNA-HindIII digest size marker; lane y: PhiX174 RF DNA-999111 digest size marker. Figure 6. 53 Southern analyses of plasmid DNAs containing MRASl and MRAS3 DNA fragments, and bacteriophage DNA containing an MRASZ DNA fragment. Panel A, an MRASl recombinant plasmid was digested with 9991 and HindIII and a 310 bp fragment was generated. Panel B, an MRASZ phage clone was digested with 999RI and 99mHI and a DNA fragment slightly larger than 600 bp was generated. Panel C, a 460 bp §99RI and HindIII restriction fragment was generated by digest MRAS3 recombinant plasmid. These restriction digests were resolved on 1% agarose gel and photographed by transillumination with short wave UV-light. Photographs of the gels are shown on the left in panel A, B, C. The MRASl 310 bp 5991/999RV fragment, the MRASZ 600 bp E99RI/999HI fragment and the MRAS3 450 bp 999I/HindIII fragment were used to prepare DNA probes and hybridized to individual blots in panel A, B, C respectively. The nitrocellulose filters were washed under low stringency condition (0.1XSSC/0.1%SDS at 40°C for 60 minutes). The washed filters were exposed to X—ray film without an intensifier for 2 hours at rm.temp. The autoradiographs are shown on the right side of each panel. 54 A 1 2 3 hp 2030 1353 m __./ :1078\\:. \603/ 31 O/— “ “ 320 2030 ....\ 1078¥ 72/": 03/ B123 C 23 ”P /2320 15.3... 2030 ’ / 1353K /1078~\ \ ~ . \T\:;:// ' 310/ 55 co-migration of restriction endonucleases with the DNA restriction fragment resulting in gel retardation. In order to ensure this restriction fragment was the subclone from MRASZ gene, Southern analysis was conducted on DNA transferred from this gel to a nitrocellulose filter using the 580 bp EEQII/999HI fragment as the probe. This restriction fragment of the MRASZ gene (Figure 1) hybridized to the E99RI/999HI fragment released by digestion of the M13 clone (Figure 6b). The recombinant bacteriophage M13mp18 containing the subcloned 22911/99mHI restriction fragment of MRASZ was named MRASZ-O. Transformant clones containing the subcloned SacI/HindIII fragment of the MRAS3 gene were analyzed using a procedure similar to that described for the MRASl-l subclone. Three different double restriction endonuclease digestions conducted on the recombinant plasmids demonstrated that all clones contained the expected restriction fragment (Figure 5). One transformant was selected to prepare large quantities of the recombinant plasmid. The resulting recombinant plasmid was analyzed by digestion with E99RI and fi199III endonucleases. A 460 bp 999RI/fl199III restriction fragment (E99RI site located in the polylinker region of plasmid) was generated (Figure 6c). In Southern blot analysis, this 460 bp EcoRI/HindIII restriction fragment hybridized with a DNA probe (Figure 6c) 56 prepared from the 999I/fl199III restriction fragment of MRAS3 gene demonstrating that the 450 bp 999I/fl199III restriction fragment (Figure 1) had been ligated into plasmid pUC19 and transformed into 91 9911 DH5a. This plasmid containing the subclone of the MRASB gene was named MRAS3-21. Determining the Similarity between the Three Mucor RAS Gene Subclones-MRASi-l, MRASZ-o and MRAS3-21 In order to minimize the risk of cross-hybridization in the analysis of MRAS gene expression using MRASl-l, MRAS2-0 and MRA83-21 as gene-specific probes, it was necessary to examine the similarity of these three DNA restriction fragments in advance. The subcloned restriction fragments from MRASl-l, MRASZ-O and MRAS3-21 were purified by agarose gel electrophoresis followed by electroelution. The purified DNA fragments were resolved in triplicate on a agarose gel and subjected to Southern hybridization analysis using MRASl-l, MRASZ-O and MRAS3-21 as DNA probes (Figure 7). The MRASl-1 and MRAS3-21 subclones cross-hybridized to each other to a limited extent. The hybridization signals were analyzed by scanning the autoradiograph with a densitometer. The strength of cross-hybridization of MRASl-l to MRAS3-21 was only about 12% of the specific binding of the MRASl-l probe to itself: ie. the strength of MRAS3-21 Figure 7. 57 Nucleotide sequence similarity between the three Mucor ras gene subclones. The subcloned restriction fragments from MRASl-l (sample 1), MRASZ-O (sample 2) and MRAS3-21 (sample 3) were purified by gel electrophoresis and resolved in triplicate on an agarose gel and subjected to Southern blot hybridization using MRASl-l (panel A), MRASZ-O (panel B) and MRAS3-21 (panel C) as DNA probes. Nitrocellulose filters were washed under high stringency conditions (0.1XSSC/0.1%SDS solution 65°C for 60 minutes) and then exposed to X-ray film for 2 hours without intensifier at rm temp. Autoradiographs resulting from these Southern analyses are shown. 58 59 specific binding was more than 8 fold greater than cross- hybridization to MRASl-l (Table 1). According to the nucleotide sequence data, the hypervariable region of the MRAS genes is located in exon V of each gene. The MRASl-l restriction fragment contains the entire region of exon IV and approximately half of exon V. The MRA83—21 restriction fragment includes exon III, exon IV and most of exon V. The cross-hybridization of these two restriction fragments is likely to be due to the higher similarity between MRASl-l and MRAS3-21 in exon IV. Based on computer assisted dot matrix analysis (80% stringency) data, a 50 bp fragment of MRASl-l and a 90 bp fragment of MRASB-Zl showed the least similarity between each other. Both fragments were located within exon V of each gene. As expected, MRASZ-O did not cross-hybridize to either MRASl-l or MRA83-21 because this restriction fragment contains only exon V and a 3' segment of flanking sequence. The Southern hybridization data and dot matrix data showed good agreement and suggested that the MRAS probes would be useful for preliminary analyses of MRAS transcript levels. It was believed that these probes would not cause significant artifacts in detecting MRAS gene transcript levels and would differentiate among transcripts of the three MRAS genes. Table 1. 60 Analysis of cross-hybridization of MRAS probes. Columns contain data for DNA probes used for hybridization. Column A, MRASl-l DNA fragment; probe 1. Column B, MRASZ-O DNA fragment; probe 2 and column C, MRA83-21 DNA fragment; probe 3. Numerical values represent integrated area of absorbance peak measured in square pixels where one pixel represents a unit information resolved by densitometer from the densitometer scan of the autoradiographs shown in figure 7. Rows contain data from the target DNAs on the nitrocellulose filter DNA samples. Row 1, target DNA fragment MRASl-l; Row 2, MRASZ-O as target DNA: and Row 3, MRASB-Zl as target DNA. 1 3842 - 495 2 - 3741 - 3 574 - 4019 61 Northern Analysis of M. r9cemosus RAS Transcript levels with DNA Probes MRASl-l, MRASZ-O and MRASB-Zl were used to prepare hybridization probes to analyze the Mucor ras gene transcripts in this study. The DNA probes MRASl-l, MRAsz-O and MRAS3-21 were labeled with [04”P]dGTP to a high specific activity (1-5 x 108 cpm/pg DNA). These three DNA probes were used to analyze the accumulated levels of ras mRNA from several cell morphologies of M. r9cem999s (Figure 8) to determine whether there was a morphology- related change in expression of M9999 ras genes. mRNAs were purified from immature spores and sporangiophores (sporulating cells), sporangiospores, germinating spores (1 hour and 4 hours incubation), germlings (12 hours germination), germling-to-yeast transition cells (1 hour and 3 hours), yeast cells, and yeast cells which has been induced to undergo morphogenesis to hyphae (see materials and methods). Polyadenylated RNA was isolated from these different morphologies of M. ecemosus, resolved on formaldehyde-agarose gels and transferred to nitrocellulose filters. The probes described above were used to hybridize to the RNA samples on filters under high stringency conditions. 62 Figure 8. Morphologies of Mucor :9cem99us selected for isolation of poly(A) -RNA to be used in Northern analysis. Solid segments represent aerobic growth conditions and hatched segments represent anaerobic processes. 1, sporangiospores; 2, one hour-old germinating spores: 3, four hour-old germinating spores; 4, twelve hour-old germlings; 5, germling-to-yeast one hour transition cells; 6, germling-to—yeast three hours transition cells; 7, yeast cells: 8, yeast-to-hyphae transition cells, and 9, sporangiophores, sporangia and immature spores (sporulatintg cells). 64 The pattern of transcript accumulation detected by each probe was first quantitated visually and differed significantly between the different probes. The MRASl-l probe hybridized at detectable levels to transcripts from several different morphologies of M9999 mRNA (Figure 9). The MRASl gene appeared to be expressed specifically in germinating spores (4 hours of incubation), germlings, yeast-to-hyphae transition cells and sporulating cells. The size of the MRASl transcript detected in these morphologies was 1.3 Kb. The transcript which hybridized to MRA82-0 probe was found in yeast and yeast-to-hyphae cells and was about 2.3 Kb in size (Figure 9). The MRAS3-21 probe hybridized only to a 1.3 Kb transcript in sporulating cells (Figure 9). The MRAS3 transcript detected in sporulating cells is not likely to be due to cross-hybridization of MRA83-21 probe to the MRASl transcript because the pattern of transcript accumulation of MRASl and MRASB is different. Moreover, the accumulated level of MRASB transcript in sporulating cells is similar to that of the MRASI transcript (Table 2), which would most likely not be the case if the signal was due to cross hybridization. In a control experiment, the identical blots were hybridized with a TEF-l probe (Figure 9). This gene encodes elongation factor-la in M. 999999999 and is expressed at constant levels in 4 of the morphologies tested here (ie. Figure 9. 65 Northern analysis of M9 gacemosus poly(AQ+-RNA with MRAS DNA probes. Poly(Ao -RNA was isolated from several different morphologies of Mucor cells, resolved on formaldehyde-agarose gels, and blotted to nitrocellulose filters. Lane 1, polyadenylated RNA from sporangiospore: Lane 2, germinating spores (1 hour); Lane 3, germinating spores (4 hours); Lane 4, germlings: Lane 5, germling-to-yeast cells (1 hour); Lane 6, germling-to-yeast cells (3 hours); Lane 7, yeast cells; Lane 8, yeast-to-hyphae cells, and Lane 9, sporulating cells. MRAS DNA probes were radiolabelled and used to probe Northern blots: Panel A, MRASl-l probe; Panel B, MRASZ-O probe: Panel C, MRAS3-21 probe. Panel D was probed with a TEF-l probe. Shown are autoradiographs from each hybridization reaction. Nitrocellulose filters were washed 0.1XSSC/0.1%SDS solution at 65°C for 60 minutes (high stringency condition). Blots A, B and C were exposed to X-ray film with an intensifier at -70°C for 72 hours. Blot D was exposed to X-ray film with an intensifier at -70°C for 16 hours. 66 67 sporangiospores, germlings, yeast, yeast-to-hyphae 3 hours transition cells, Linz and Sypherd 1987). In the control experiment, the TEF-l probe hybridized at significant levels to a 1.5 Kb transcript in germinating cells (1 and 4 hours), germlings, yeast cells, yeast-to-hyphae transition cells and sporulating cell mRNAs and at reduced levels with the same size transcript germling-to-yeast (3 hours of incubation) transition cells. No transcript was detected in sporangiospores and germling-to-yeast (1 hour) transition cells with the TEF-l probe. This suggests that either no TEF-l transcript is present at this stage of growth or that the mRNA in this sample was degraded. Since no transcript was detected in RNA from sporangiospores or germling-to- yeast (1 hour) transition cells using the MRAS probes, this may be due to artifacts arising from absence of detectable levels of mRNA in these samples.) The control experiment suggests that the quantities of mRNAs from each morphological stage used in the Northern analysis of MRAS genes transcript were not constant (Table 2), especially in the sample from germling-to-hyphae (1 hour) cells. Further work is needed to normalize mRNA quantities for each sample using the accumulated levels of the TEF-l transcript as the control level. Table 2. 68 Analysis of Northern hybridization of M999; RNA with DNA probes. Columns contain data from M9999 poly(AQ'-RNA samples isolated from different morphologies of M999; cells used in Northern analysis of figure 9. Column 1, sporangiospore: 2, germination spores (1 hour): 3, germination spores (4 hours); 4, germlings: 5, germling-to-yeast (1 hour): 6, germling-to-yeast (3 hour): 7, yeast cells: 8., yeast-to-hyphae and 9, sporulating cells. Numerical values represent integrated area of absorbance peak measured in square pixels where one pixel represents a unit of information resolved by densitometer peak from the densitometer scan of autoradiographs shown in figure 9. Rows contain data from the DNA probes used for Northern hybridization analysis of M. :a99m059s RAS transcript levels. 5177 7268 5766 4304 3487 16437 7355 - - 1487 2897 - - 7920 3704 - - - - - - 295 2737 - - - 3271 DISCUSSION It has been proposed that ras genes may play a fundamental role in basic cellular functions in eukaryotic cells. Two cellular functions which have been experimentally linked to ras expression are cell differentiation and cell proliferation. We are studying M. 999999999 as a simple model system for cell proliferation and differentiation in eukaryotes. Three ras genes have been cloned previously from M999; 199999999. We hypothesized that if one or more ras genes are involved in regulation of morphogenesis (cell differentiation) in this organism, the expression levels of the genes may correlate with changes in morphology of M. :99emos9s. This project focused on the preliminary detection of the expression levels of three M999: ras transcripts, MRASl, MRASZ and MRA83 using DNA probes. DNA restriction fragments containing a portion of the 3' end hypervariable region (exon V) and additional sequences were subcloned from each MRAS gene. The similarity among these three MRAS gene subclones was tested by Southern hybridization and computer assisted dot matrix analyses. Both types of analysis demonstrated that MRASl-l 69 70 and MRAS3-21 did share some sequence similarity. However, the cross-hybridization between MRASl-l and MRAS3-21 probes was only about 12% to 15% of the specific hybridization level. Significant similarity between MRASZ-O and MRASl-1 or MRA83-21 was not detected. Based on these data, we concluded that these three subclones were useful in preparing DNA probes for preliminary detection and quantitation of MRAS transcripts. Polyadenylated mRNA from eight morphologies of M9999 cells were analyzed. The preliminary data from this study suggested that MRASl and MRAS3 genes were expressed in a morphology-specific pattern because the accumulated transcript levels of these two MRAS genes correlated with the morphological changes of M999; 999999999. However, the MRASZ transcript, which was detected in yeast and yeast-to- hyphae transition cells was still in doubt because this probe contained DNA sequences downstream from the MRASZ gene (flanking sequence). At least two ras genes, MRASl and MRASB, therefore may be involved in regulation of M9999 morphogenesis. In a recent study, the quantities of polyadenylated mRNA purified from several cell morphologies of M. 999999999 have been normalized (by TEF-l transcript levels as control) and used to repeat this Northern blot analysis (J. Linz, 71 unpublished data). DNA restriction fragments containing a portion of exon V and flanking sequences of MRASl and MRAS3 genes were used to prepare DNA probes. In addition, a third fragment located totally within the predicted open reading frame (protein coding sequence) of MRASZ was used (Figure 10). The results revealed that the MRAsz gene was not expressed at detectable levels in any of the morphologies of M999; cells. The previous experiment which showed a putative 2.3 Kb (figure 9) MRASZ transcript in yeast and yeast-to-hyphae cells was due to the hybridization of flanking sequence of MRASZ-O probe to M999; mRNA. The expression data and nucleotide sequence data suggested that the MRASZ gene may be a pseudogene (ie. totally inactive), is expressed at extremely low levels, or is expressed at times not sampled in this study. DNA probes prepared from the MRASl gene hybridized to a 1.3 Kb mRNA in all morphologies of M999; cells and the accumulated transcript level varied considerably. MRAS3 DNA probes which hybridized strongly to the mRNA of sporulating cells, hybridized to mRNA of germlings moderately, and slightly to yeasts in the more refined analysis. These new data confirm the previous result that two MRAS genes are differentially expressed. These MRAS transcripts are accumulated in a morphology-related pattern. Figure 10. 72 DNA probes used for the refined Northern analysis of MRAS transcripts. Probe 1', the Econ/Ecogv restriction fragment (3' E9931 site not shown)containing part of the exon V of MRASl gene and 3' flanking sequence; Probe 2', the EchV/EyuI; internal restriction fragment of MRASZ gene, and Probe 3', the 9199111/9199111 restriction fragment (3' M1nd111 site not shown) containing a portion of exon V of MRASB gene and 3' flanking sequence. 73 HRASI IV III II HHom >moou Hana HOAN Haom Hflnm Hafiz Probe 1' HRASZ IV III I1 HHHakd HHSQd HH§>m Hanna 4m5¢m Honz >moom Heua HHUGwm HHHskd Probe 2’ HRASB HHHUflHm Hunm IV Hooz HH5>m III Hoem HHdm II Probe 3' 74 The results of this study indicated that expression of M999; ras genes may be regulated during cellular morphogenesis. Changes in the requirement for individual ras protein may result in the alteration of expression of each M999; ras gene. However, investigation of the biological function of M999; ras genes is necessary in future studies. One approach to discovery of the normal biological function of the MRAS genes is to qualitatively and quantitatively alter MRAS gene products and observe changes in cell growth and development. Expression of antisense mRNA or overexpression of M9999 ras genes may be useful tools to explore the correlation of MRAS gene activity and the physiological response of M999; cells. These tools may provide a crude mechanism to artificially regulate the ras protein level and activity in the cell. Due to the correlation of signal transduction and morphogenesis in Mucor, the effects of ras gene expression on cAMP and phosphatidylinositol phosphate (PI) metabolism, two important secondary messengers, may also be observed. The identity of effector proteins, receptor proteins and other components of a putative signal transduction pathway with which MRAS proteins may interact will be helpful in clarifying the function of individual M9999 ras proteins. Monoclonal antibodies, specific to individual M999; ras proteins, may also be useful to determine the location of 75 ras proteins in M9co; cells and to confirm the biological function of M9co: ras proteins by blocking these activities in vitro. Normal mechanisms for control of M999; ras gene transcription are also important to study. The alteration in level of transcript may be due to either the rate of transcription or rate of mRNA degradation. To investigate this problem, the identification of promoter regulatory regions, and regulatory proteins involved in MRAS gene expression would be helpful to clarify the regulatory mechanism at the transcriptional level. The exploration of transport and processing mechanisms of ras hnRNA may be useful in understanding the alterations in accumulated transcript level and RNA degradation. 76 CONCLUSION The finding of three ras gene homologues in the filamentous fungus M9999 999999999 led to these studies designed to explore the correlation of MRAS gene expression and cellular morphogenesis in this organism. This study showed that at least two MRAS genes were expressed differentially and that the accumulated transcript levels of the MRASl and MRAS3 genes varied considerably in a morphology-related pattern. These experimental data confirm one part of the original research hypothesis that the expression levels of the MRAS genes may correlate with changes of M9999 cell morphology if one or more MRAS genes are involved in regulation of morphogenesis. 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