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MICRO-MANIPULATION OF CHICKEN CHROMOSOMES AND DEVELOPMENT OF CHROMOSOME-SPECIFIC DNA LIBRARIES. F. Abel Ponce de Leon 1, Yukui Li 1, Sakthikumar Ambady 1, David Burke 1, James Bitgood 2 and James Robl 1. 1Dept. of Veterinary MA. 01003-6410. and Animal Sciences, University of Massachusetts, Amherst, 2Dept. of Poultry Sciences, University of Wisconsin, Madison, WI 53706 INTRODUCTION The genome of Gallus domesticus is about 1/3 of the mammalian genome, or approximately 1.1 to 1.4 pg of DNA per haploid cell. The chicken chromosome complement is divided in two distinct groups of 10 macrochromosomes and 29 microchromosomes, for a total of 39 chromosomes per haploid genome. This translates to having about 65 % of the genome contained in the macrochromosomes and 35 % in the microchromosomes. Chromosome 1 contains about 17 % of the total DNA, while the Z chromosome contains about 8 % (Bitgood and Shoffner, 1990; Fechheimer, 1990). This cytogenetic observation correlates well with the fact that approximately 90 % of all genes, so far mapped, have been assigned to macrochromosomes (Bitgood and Somes, 1990). The first chicken linkage map was published by Hutt (1936) and contained 18 loci distributed in five linkage groups. At present the map consists of over 200 loci, and over 20 linkage groups and some gene assignments to microchromosomes (Levin et al. 1994). During the last 5 decades the development of the chicken map has been approached mainly by family segregation studies and linkage anchoring based on chromosomal rearrangement break points. Other approaches such as somatic cell hybrids (Kao, 1973) and chromosomal isotopic in situ hybridization have also been utilized but have not yet made significant contributions to the chicken genome map. Other approaches have been implemented with the objective to produce a 10 cM chicken gene map within the next five to ten years. These include the development of two reference mapping populations (Bumstead et al , 1992, Crittenden et al, 1993). These populations are enabling the construction of genetic ' linkage maps based on molecular genetic markers. Likewise, the utilization of chromosomal fluorescent in-situ hybridization (FISH) is providing information about anchor loci to facilitate chromosomal assignments of all available linkage groups (Ponce de Leon et al., 1992), hence allowing the development of cloning strategies for important regions of the genome. FISH will help in developing a map of regularly spaced polymorphic loci by providing quick and accurate localization of polymorphic genomic sequences. Partitioning of the chicken genome into small units to concentrate mapping efforts in small regions of the genome becomes an important task, particularly when over 65 % or the genome is compartmentalized into very few chromosomes. Fragmenting the genome by individual chromosomes and construction of chromosome-specific DNA libraries is therefore a logical approach. We have microisolated and microcloned two chicken macrochromosomes. Our work was 18 facilitated by the fact that chicken macrochromosomes are identified by morphology which allowed the microisolation of 10 copies of the chromosome of interest. This small number of copies yielded about 0.7 to 1.0 pg of DNA as the starting cloning material. We describe here the general strategy for chromosome microisolation and microcloning, as well as the use of the strategy for the identification and FISH assignment of large insert sequences. MICROCLONING STRATEGY Metaphase preparations. Conventional fibroblast cell cultures were carried out as previously described (Ponce de Leon et al. 1992). Briefly, after the hypotonic treatment of the cell suspension, chromosomes are fixed by three changes (5 min, each) of methanol:acetic acid at 9:1, 5:1, and 3:1 before placing the cells on cleaned ice cold coverslips. This mild fixation reduces depurination of DNA caused by prolonged exposure to 3:1 methanol:acetic acid fixation as it is done with conventional procedures. Microscraping. With the help of an inverted microscope and hydraulic micro manipulator, chromosomes are scraped from the surface of coverslips. After scraping is completed the scraped chromosome is picked up with a micro needle and transported to a siliconized coverslip. For each experiment ten copies of the chromosome of interest are accumulated. Sau3A I Adaptor. Two oligomers, a 28mer and a homologous 24mer designed to include a Sau3A I overhang site at its 5' end and an EcoR I restriction site close to the 3' end were prepared. Phosphorilation of the 28mer at its 5' end and annealing of the two oligomers to form the double stranded adaptor molecule have been previously described (Ponce de Leon and Robl, 1992). This adaptor was designed to be ligated to chromosomal inserts generated by the microcloning procedure. The adaptors provide a known priming site for polymerase chain reaction (PCR) amplification of chromosomal inserts (Fig 1). Microcloning. General procedures and buffer compositions have been described elsewhere (Ponce de Leon and Robl, 1992; Saunders et al. 1989). Briefly, scraped chromosomes are covered with a 1 nl drop of proteinase K buffer that is immediately protected from evaporation with a large drop of heavy mineral oil. Protein digestion is carried out for 2 hours at 37 C. The coverslip carrying the chromosomal DNA is inverted and placed in an oil chamber that is filled with heavy mineral oil. The chromosomal DNA remains in the 1 nl hanging drop. Three phenol and one chloroform:isoamylalcohol (24:1) extractions are followed by a Sau3A I (80U/ul! nuclease digestion. The preparation is incubated for 2 hours at 37C and the enzyme is inactivated at 65C for 30 min. Sau3A I adaptors, T4 ligase buffer and T4 ligase are added to the nanoliter drop and incubated overnight at 4C. The hanging drop is then taken in a micropipette and transfered to a 10 ul volume of Bgl II digestion reaction to digest the adaptor dimer molecules generated after ligation. After inactivation of the Bgl II enzyme the preparation is transferred to a 100 ul PCR reaction. This reaction includes 1 uM primer (24mer oligomer). Amplification is checked by electrophoresis in a 2% agarose gel. 19 CHROMOSOME PAINTING. Fluorescent in situ hybridization (FISH). The protocol as outlined by Lichter et al., (1988, 1990) and Ponce de Leon et al., (1992) was carried out. Slides containing metaphase spreads were incubated for exactly two minutes, in 70% deionized formamide, 2X Sodium saline citrate (2X SSC) at 70°C. Slides were immediately dehydrated in 70%, 90%, 100% ice cold ethanol for five minutes each. The pool of amplified chromosomal inserts (chromosome cocktail) was labeled by nick translation using biotin-16-dUTP which substitutes dTTP in the standard nick translation reaction mixture. Since genomic DNA contains repetitive sequences that are common to other chromosomes, we prepared competitor DNA in order to prevent hybridization signals originating from repetitive sequences. For this chicken genomic DNA obtained from fibroblasts or fetal tissue and salmon sperm DNA were digested with DNAse I to produce fragments with equivalent size distribution as the nick translated DNA probe. _ Between 30 and 60 ng of labeled probe DNA were mixed with 2 to 4 ug. of competitor chicken DNA and enough salmon sperm DNA to yield a total of 10 ug of DNA in 10 ul of hybridization solution. The DNA mixture was denatured at 75°C for 5 minutes, followed by incubation at 37°C for 10 to 15 min to allow preannealing of repetitive DNA sequences. The denatured and preannealed DNA mixture was then applied to prewarmed (42°C) slides with denatured chromosome preparations. Slides were placed in a humidified chamber and incubated overnight at 37uC. Following the hybridization procedure, slides were washed in 50% formamide (3 times for 5 min at 420C) and in 0.1X SSC (3 times for 5 min at 60°C). Slides were then incubated in blocking solution (3% BSA, 4X SSC) for 30 rain at 37°C. Detection of the probe was accomplished by incubating the slides, in the dark, for 30 min at 37°C with fluorescein isothiocyanate (FITC)-conjugated avidin DCS (5 ug/ml, Vector Laboratories) which is made up in 4X SSC, 0.1% Tween 20, 1% BSA. After incubation, the excess detection solution was washed (in the dark) in 4X SSC, 0.1% Tween 20, 3 times for 5 min at 42°C. Slides were counterstained in a 200 ng/ml propidium iodide solution. After washing, slides were mounted in antifade p-phenylenediamine free base (Sigma) solution (PPD-11). Microscopy. Slides were screened under a microscope (Zeiss, Axioskop) equipped for epifluorescence microscopy. Propidium iodide staining and FITC FISH signals will be detected with 546/590 and 450/520 excitation/barrier filter combinations, respectively. A SIT 66 video camera, that is attached to the microscope, was used to capture images that were digitized by an Image 1/AT software (Universal Imaging Inc.). Images were stored in 44 mb disk cartridges (Bernoulli). Photomicrographs of digitized images were prepared with a color video printer (Sony, model UP 5000). CHROMOSOME SPECIFIC DNA LIBRARIES. A fraction of the amplified chromosome specific inserts was digested with Sau 3A I according to the manufacturer' instructions. This generated Sau 3A I sticky ends that were ligated to a comparable site of the Bam HI Lambda Zap II vector (Stratagene). After infection of NM554 cells and plating recombinant clones can be identified by white/blue screening. Libraries have been amplified and stored as described in the manufacturer's protocols. PCR amplification of individual 2O chromosomal inserts can be done using the RNA polymerase flanking sites of the vector. CHROMOSOME T3 and T7 priming 1. A chromosome 1 library of small inserts has been generated and is being used for identification of clones containing microsatellite sequences. The chromosome cocktail has been used, both as a painting probe and as a probe for identification of chromosome 1 cqspaid clones. This latter use of the chromosome cocktail was accomplished by "_':P end labeling of the chromosome 1 cocktail and blocking of repetitsve DNA sequences by annealing of Cot 1 unlabeled chicken DNA (Fig 2). Therefore, unique over represented sequences and/or middle repetitive sequences were used for cosmid screening. One hundred positive cosrnid clones were isolated, rescreened, and FISH assigned in pools of five per experiment. Seventy-two cosmid clones were chromosome 1 positive and 20 of these clones have been assigned to specific sites on chromosome 1 (Fig 3). Z CHROMOSOME. The Z chromosome has been microcloned as described before. We have use the Z chromosome cocktail as a painting probe to asses its origin and purity. This is important because contamination with DNA Originating from other chromosomes could compromise the purity of the preparation. Contamination results from erroneous chromosome identification before the scraping and/or accidental scraping of pieces of other chromosomes surrounding the chromosome being scraped. The Z chromosome painting probe has been used to analyzed the NM 7659 t(Z;1) chromosome rearrangement (Zartman, 1973). Preliminary observations resulting from the analysis of the FISH signals obtained with the Z chromosome cocktail indicate that the break points for the reciprocal translocation occurred at Zp23 and lq13. The Zp24 band has been translocated to lqll. This would indicate the translocated lq lost band lq12. This loss of chromatin might be one of the reasons or the most important reason why birds homozygous for the translocation have not been generated in spite of successive efforts (Bitgood, personal observation). Our intention is to use the NM 7659 t(Z;1) rearrangement, and other rearrangements that include the Z chromosome, to confirm the localization of the ev 21 locus. This locus was reported to be localized at Zp24 (Lakshmanan et al. 1992), while the current linkage maps report it to be more proximal to the centromere region of the Z chromosome (Bitgood and Somes 1990). This approach will initiate the process of integration of classical, molecular and physical maps. REFERENCES. Bitgood J.J. and R.N.Shoffner RN, Cytology and cytogenetics. and genetics, 1st ed (Crawford RD, ed). New York: 1990. In: Poultry breeding Elsevier; 401-427. Bitgood J.J. and R.G. Somes. Linkage relationships and gene mapping. In: Poultry breeding and genetics, 1st ed (Crawford RD, ed). New York: Elsevier; 469495. 1990. 21 / "' Bumstead, N. and J. Palyga. A preliminary Genomics 13:690-697. 1992. linkage map of the chicken genome. Crittenden, L.B., L. Provencher, L. Santangelo, I. Levin, H. Abplanalp, R.W. Briles, W.E. Briles and J.B. Dodgson. Characterization of a Red Jungle Fowl by White Leghorn backcross reference population for molecular mapping of the chicken genome. Poultry Science 72:334-348. 1993. Fechheimer N.S. Chromosomes of Chickens. In: Domestic animal cytogenetics, 1st ed (McFeely RA, ed). San Diego, California: Academic Press Inc; 170-207. 1990. Hutt, F.B. Genetics of the fowl. VI. A tentative chromosome map. Neue Forsch. Tierzucht Abstam. (Duerst Festschrift) pp 105-112. 1936. Kao, F. T. Identification of chicken chromosomes in cell hybrids formed between chick erythrocytes and adenine-requiring mutants of Chinese hamster cells. Proc. Natl. Acad. Sci. USA 70:2893-2898. 1973. Lakshmanan, N., F.A. Ponce de Leon, J.R. Smyth and E.J. Smith."Chromosomal assignment of the ev21 locus in chicken using fluorescent in situ suppression hybridization (FISH)". Proceedings. 10th European Colloquium on Cytogenetics of Domestic Animals. Utrecht University. The Netherlands. p. 10, 1992. Levin, I., L. Santangelo, H. Cheng, L.B. Crittenden and J.B. Dodgson. An Autosomal Genetic Linkage Map of the Chicken. J. of Heredity 85:79-85. 1994. Lichter, P., T. Cremer, J. Borden, L. Manuelidis and D.C. Ward. Delineation of individual human chromosomes in metaphase and interphase cells by in situ suppression hybridization using recombinant DNA libraries. Hum. Genet. 80:224-234. 1988. Lichter, P., C.C. Tang, K. Call, G. Hermanson, G.A. Evans, D. Housman and D.C. Ward. High-resolution mapping of human chromosome 11 by in situ hybridization with cosmid clones. Science 247:64-69. 1990. Ponce de Leon, F.A., Y. Li and E.J. Smith. Reassignment of the evl locus by high resolution chromosomal in situ localization. Poultry Science 70(1):95, 1991. Ponce de Leon FA, Li Y, Weng Z. Early and late replicative chromosomal patterns of Gallus domesticus. J. of Heredity 82:36-42. 1992. banding Ponce de Leon, F.A. and J.M. Robl. Microisolation and Microcloning Of the Bovine X-chromosome. 23rd International Conference on Animal Genetics. ISAG Interlaken, Switzerland, University of Berne, p 62, 1992. Saunders, R.D.C., D.M. Glover, M. Ashburner, I. Siden-Kiamos, C. Louis, M. Monastirioti, C. Savakis and F. Kafatos. PCR amplification of DNA microdissected from a single chromosome band: a comparison with conventional microcloning. Nucleic Acids Research 17:9027-9037. 1989. Zartman, D.L. Location of the pea comb gene. Poultry Science 52:1455-1462. 22 23 FIG. 2 Strategy for Screening and Identification of Chicken Chromosome 1 Cosmid Clones ® End Labeling '_ ® ® ® ® ® ® Chromosome-cocktail Probe " ® -. 1 T ® ® ® ® ® ® Denaturation ® ® Cot 1 DNA ® Reanneling Cosmid Library Screening 24 -FIG. 3 FISH Assignments of Chromosome 1 :osmid Clones UMA0071 I 56 UMA0014 UMA0037 2 4 2. _0004 I UMA0028 3 5 _0045 UMA0010 UMA0009 UMA0074 UMA0032 UMA0030 UMA0036 UMA0027 UMA0024 /_ 12 34 .s UMA0082 UMA0008 UMA0073 2 UMA0021 UMA0020 4 [ 25 1 1 2 3 4 I Question: Chris Tuggle, ISU Have you used a newly exported technique called PCR-in situ (Troyer, et al, 1994)? Response: A. Ponce de Leon No, DISC-PCR as the technique has been named, has been recently developed. Even though it allows assignment of small fragments of DNA by PCR amplification directly from chromosomes, it also requires the observation of a very large number of metaphase plates and statistical analysis of signals observed to determine exact localization. The technique does not allow direct identification of chromosomes because, as of now, chromosomal banding pattern can not simultaneously be visualized. However, it is a promising technique and other improvements might be coming later. Question: J. Zhu 1) Did labeling more than one nucleotide increase sensitivity of staining? 2) How many bases should be apart to be able to be recognized by antibodies? Response: A. Ponce de Leon 1) Using more than one biofinalated nucleotide increased the sensitivity of the FITC signal. Best results were obtained with the use of Biotin-16-dNTP and Biotin-14dATP. 2) The system we use requires AVIDIN-FITC and there is no stereochemical difficulty with regard to distance between biofinalated nucleotides. The 16 and 14 carbon chain to which Biotin is attached reduces steriochemical interference in a very significant way. 26 Comment: L. Crittenden Seven Chromosome cosmid clones were sent to East Lansing by Dr. Ponce de Leon. These were used as probes for molecular genetic mapping. One was a single locus probe and allowed us to assign a linkage group to the long arm of chromosome 1 for the first time, thus this approach can be used to assign linkage groups from the molecular genetic map to chromosomes. Speaker: A. Ponce de Leon Question: David Vaske With the microscoping/microisolation techniques, how many of the chicken chromosomes are large enough to be isolated (and identified) for production of the chromosome specific libraries? Response: A. Ponce de Leon Eight pairs ofautosomes and the sex chromosomes will be in time, microisolated for the generation of chromosome - specific library. The remaining chromosome are called micro-chromosomes and contain 35% of the chicken genome. The best approach would be to micro-isolate all micro-chromosomes together to make a micro-chromosome specific DNA library. 27 Question: E. Smith For your primary library wouldn't a PI or BAC library (probably not a YAC for now) bc better? and cosmids only for say, sub cloning? Response: A. Ponce de Leon Yes, it would be, however costs to produce a PI or a BAC library are also greater than for a cosmid library. In an environment where research dollars are tight having a cosmid library is good enough. 28