Occupancy of the majority of DNA in the chicken W chromosome by bent-repetitive sequences

Another family of repetitive sequences, designated the EcoRI family, was found in the DNA of the chicken W chromosome by hybridization with the W chromosome-specific XhoI family probe under conditions of low stringency. A 1.2 kb EcoRI fragment, the major repeating unit of the family, was cloned and sequenced. The 1.2 kb unit showed an overall sequence similarity of about 68% to the 0.7 kb XhoI family repeating unit and it consisted of tandem repeats of average length 21 bp, most of which contained (A)_3–5 and (T)_3–4 clusters separated by 6–8 G+C-rich sequences. These features and its behavior as a strongly bent molecule in solution were very similar to those found for other W chromosome-specific repetitive sequences in the order Galliformes: XhoI family of chicken, PstI family of turkey and TaqI family of pheasant. The cloned 1.2 kb unit contained 78 CpG dinucleotide sequences and those that were in HapII, HhaI and BstUI sites were shown to be extensively methylated in the genomic DNA. Repetition frequencies of the 1.2 kb unit among the female population of chicken fell into high- and low-level classes, which accounted for about 30% and 10%, respectively, of the DNa in the W chromosome. Thus, 70% to 90% of the DNA in the chicken W chromosome was shown to be occupied by bent-repetitive sequences. The EcoRI and XhoI family sequences were not intermingled over the short range but each family formed a unique domain ranging from one to several million base pairs.by H.C. Macgregor     

Purification and characterization of W-protein. A DNA-binding protein showing high affinity for the W chromosome-specific repetitive DNA sequences of chicken

A protein component, which binds with high affinity to the W chromosome-specific XhoI family repetitive DNA of chicken (Tone, M., Sakaki, Y., Hashiguchi, T., and Mizuno, S. (1984) Chromosoma (Berl.) 89, 228-237), was detected in the 0.35 M NaCl extract of the female chicken liver nuclei. This protein, designated as W-protein, was substantially purified by phosphocellulose, hydroxyapatite, and DEAE-Toyopearl column chromatography. Molecular weight of W-protein was estimated to be about 72,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, but it seems to form multimeric structure having apparent molecular weight of about 2.3 X 10(6) under nondenaturing conditions. W-Protein binds strongly to both 0.7- and 1.1-kb repeating units of the XhoI family, both of which show curved DNA characteristics, and weakly to the AATAT-satellite sequence of Drosophila melanogaster. Stable binding of W-protein requires greater than or equal to 300 base pairs of the 0.7-kilobases sequence, or more than 14 tandem repeats of the 21-base pair internal repeating unit of the 0.7-kilobase sequence. DNA footprint analysis and effects of some DNA-binding compounds suggest that the DNA double helix wraps around W-protein or its multimeric form contacting through A-T-rich minor grooves. A possible role of W-protein in the formation of W heterochromatic body is discussed.     

Distribution of XhoI and EcoRI family repetitive DNA sequences into separate domains in the chicken W chromosome

Fluorescence in situ hybridization using as probes three biotinylated or digoxigenin-labeled chicken W chromosome-specific repeating DNA units (0.7 and 1.1 kb XhoI family and 1.2 kb EcoRI family units) suggested that a large fraction of one arm of the W chromosome was occupied by the EcoRI family sequences and that pericentromeric regions were widely occupied by the XhoI family sequences. A minor fraction of the EcoRI family was also present in a narrow region in the proximal half of the other arm. There was a region in the distal half of the latter arm where sequences from neither family hybridized. Evolutionary aspects of the presence of different domains occupied by different repetitive families and the significance of the unhybridized distal region are discussed.by H.C. Macgregor     

Nucleotide sequences and unusual electrophoretic behavior of the W chromosome-specific repeating DNA units of the domestic fowl,Gallus gallus domesticus

Nucleotide sequences of three independently cloned repeating units of the W chromosome-specific repettive DNA sequences (XhoI family) of the chicken were determined. All three units are 717 bp long with XhoI sites at both ends. There are only 21 sites out of 717 bases where a single base change occurs in one of the three clones. Each of these repeating units consists of 34 tandem repeats of about 21 bp. Sequences of some members of these internal repeats are not well conserved, but the majority of the repeats are characterized by the presence of (A)_3–5 and (T)_3–5 clusters separated by 6–7 relatively G+C-rich base pairs. One striking feature of the cloned 717 bp repeating units is that they migrate unusually slowly on electrophoresis in polyacrylamide gels. The same feature is also shown by a genomic population of the 0.7 kb repeating units recovered from XhoI digests of the genomic DNA of the female chicken. This anomalous behavior is attributed to the occurrence of DNA curvatures because of the above sequence characteristics and partial recovery of the electrophoretic mobility in the presence of distamycin A. Another feature of the 717 bp repeating unit is the presence of 438 and 159 nucleotide-long open reading frames (ORFs) at each end of the unit. A possible function of the XhoI family sequences in the heterochromatization of the W chromosome and the significance of the ORFs are discussed.     

Demonstration of W chromosome-specific repetitive DNA sequences in the domestic fowl,Gallus g. domesticus

Evidence is presented to demonstrate the presence of W chromosome-specific repetitive DNA sequences in the female White Leghorn chicken, Gallus g. domesticus, based on two different experimental approaches. First, ³H-labelled, female chicken DNA was hybridized with excess, unlabelled, mercurated, male DNA, and unhybridized single-stranded ³H-DNA (³H-SHU-DNA) was recovered by SH-Sepharose and hydroxyapatite column chromatography. Approximately 24% of the hybridizable ³H-SHU-DNA was female-specific and localized on the W chromosome. The second approach was to examine female-specific DNA fragments among the digests of chicken DNA with various restriction endonucleases. Among them, we found that digestion with XhoI produced two prominent female-specific bands of 0.60 kb (= kilobase pairs) and 1.1 kb. The 0.60 kb fragment was isolated and ³H-labelled by nick-translation. Female-specificity of the ³H-XhoI—0.60 kb DNA was judged to be at least 95% under the conditions of hybridization with membrane filter-bound DNA. Presence of amplified XhoI—0.60 kb DNA on the W chromosome seems to be limited to different lines of G. g. domesticus and no such repeat was detected in three species belonging to other genera in the order Galliformes and in three species belonging to other avian orders.     

Characterization of a new repetitive sequence that is enriched on microchromosomes of turkey

We cloned and characterized a new highly repetitive, species-specific DNA sequence from turkey (Meleagris gallopavo). This repeat family, which accounts for approximately 5% of the turkey genome, consists of a 41 bp repeated element that is present in tandem arrays longer than 23 kb. In situ hybridization to turkey metaphase chromosomes (2n=80) demonstrated that this sequence was located primarily on certain microchromosomes: approximately one-third of the 66 microchromosomes showed a positive signal. With respect to the macrochromosomes, hybridization was seen only in a pericentric position on nos. 2 and 3. The turkey microchromosome (TM) sequence shares motifs (alternating A_3–5 and T_3–5 clusters separated by 6–8 bp) that have been found previously in other avian tandemly repeated elements, e.g. a chicken microchromosome sequence, and W (female) chromosome-specific sequences of chicken and turkey. However, the TM sequence does not cross-hybridize under moderately stringent conditions with these other sequence. The spread and amplification of related repetitive sequence elements on microchromosomes and W chromosomes is discussed.by E.R. Schmidt     

The intragenomic polymorphism of a partially inverted repeat (PIR) in Gallus gallus domesticus, potential role of inverted repeats in satellite DNAs evolution

We report here the molecular characterization of the basic repeating unit of a novel repetitive family, partially inverted repeat (PIR), previously identified from chicken genome. This repetitive DNA family shares a close evolutionary relationship with XhoI/EcoRI repeats and chicken nuclear-membrane-associated (CNM) repeat. Sequence analyses reveal the 1430 bp basic repeating unit can be divided into two regions: the central region ( approximately 1000 bp) and the flanking region ( approximately 430 bp). Within the central region, a pair of repeats (86 bp) flanks the central core ( approximately 828 bp) in inversed orientation. Due to the tandem array feature shared by the repeating units, the inverted repeats fall between the central core and flanking region. Southern blot analyses further reveal the intragenomic polymorphism of PIR, and the molecular size of repeating units ranges from 1.1 kb to 1.6 kb. The identified monomer variants may result from multiple crossing-over events, implying the potential roles of inverted repeats in satellite DNAs variation.     

Female-specific DNA sequences in the chicken genome

Eight in silico W-specific sequences from the WASHUC1 chicken genome assembly gave female-specific PCR products using chicken DNA. Some of these fragments gave female-specific products with turkey and peacock DNA. Sequence analysis of these 8 fragments (3077 bp total) failed to detect any polymorphisms among 10 divergent chickens. In contrast, comparison of the DNA sequences of chicken with those of turkey and peacock revealed a nucleotide difference every 25 and 28 bp, respectively. Radiation hybrid mapping verified that these amplicons exist only on chromosome W. The homology of 6 W-specific fragments with chromo-helicase-DNA-binding gene and expressed sequenced tags from chicken and other species indicate that these fragments may have or have had a biological function. These fragments may be used for early sexing in commercial chicken and turkey flocks.     

Reticuloendotheliosis Virus Nucleic Acid Sequences in Cellular DNA

Reticuloendotheliosis virus 60S RNA labeled with (125)I, or reticuloendotheliosis virus complementary DNA labeled with (3)H, were hybridized to DNAs from infected chicken and pheasant cells. Most of the sequences of the viral RNA were found in the infected cell DNAs. The reticuloendotheliosis viruses, therefore, replicate through a DNA intermediate. The same labeled nucleic acids were hybridized to DNA of uninfected chicken, pheasant, quail, turkey, and duck. About 10% of the sequences of reticuloendotheliosis virus RNA were present in the DNA of uninfected chicken, pheasant, quail, and turkey. None were detected in DNA of duck. The specificity of the hybridization was shown by competition between unlabeled and (125)I-labeled viral RNAs and by determination of melting temperatures. In contrast, (125)I-labeled RNA of Rous-associated virus-O, an avian leukosis-sarcoma virus, hybridized 55% to DNA of uninfected chicken, 20% to DNA of uninfected pheasant, 15% to DNA of uninfected quail, 10% to DNA of uninfected turkey, and less than 1% to DNA of uninfected duck.     

Cloning in a cosmid vector of complete 37 kb and 25 kb ribosomal DNA repeat units from the chicken

DNA fragments of up to 40 kb containing rRNA-coding sequences have been isolated from a chicken liver DNA library prepared in the cosmid pHC79. Characterization of the cloned DNA by R-loop and restriction mapping has shown that there are two size classes of repeat unit, one of 37 kb and one of 25 kb, the larger of which is a family of units which vary slightly in size. These two classes were shown to be present in the DNA of a single chicken. The size of the internal transcribed spacer in the chicken was measured to be 4.4 kb from analysis of R-loops and heteroduplexes between chicken and Xenopus laevis rDNAs. No introns were observed in either the 18 S or the 28 S coding sequences. The number of copies of the chicken rDNA unit was measured by titration against the cloned sequences to be 202±51 per haploid genome.     

Comparative cytological study of Phasianus colchicus,Meleagris gallopavo,and Gallus domesticus

Summary Since viable intergeneric hybrids between the chicken (Gallus domesticus) and the pheasant (Phasianus colchicus) have been reported, as well as interfamilial hybrids between the chicken and the turkey (Meleagris gallopavo), the chromosome complements of the pheasant and the turkey were compared with that of the chicken. In these three species belonging to the order Galli, the Z-chromosomes appeared to be identical, while the autosomal complements of the pheasant and the turkey differed radically from that of the chicken. It was noted with some surprise that the pheasant of the family Phasianidae and the turkey of the family Meleagridae have very similar chromosome complements, at least so far as gross morphology of somatic metaphase chromosomes is concerned.This work was supported in part by grant C-5138 from the National Cancer Institute, U.S. Public Health Service, and grant C-17601 from the National Science Foundation.The authors gratefully acknowledge the generosity of Rea's Game Birds, Paramount, California, who supplied the pheasant chicks, and the McPherin Hatcheries, Sunnymead, California, who furnished the turkey chicks. The authors also appreciate the editorial assistance of'Patricia A. Ray.     

秦岭羚牛高度重复序列DNA片段的分子克隆和序列分析

羚牛（Budorcas taxicolor)属偶蹄目（Artiodactyla)、牛科（Bovidae),为我国一类大型珍贵保护动物。我们从其基因组中克隆得到若干约800bp的BamHI高度重复序列并对部分克隆进行了序列测定,发现它们显示了很高的同源性。利用其中一个单元为探针,对限制酶消化后的羚牛基因组DNA作杂交分析,发现其杂交谱带不具有个体及亚种间特异性,说明该重复序列在羚牛基因组中具有保守的分布和排列。在牛科动物中,羚牛BamHI片段与绵羊属和山羊属的相关序列具有高度同源性,而与水牛和家牛序列差异较大。这些结果为羚牛与羊亚科物种亲源关系较近的分类学观点提供了分子生物学证据。有证据表明,这些片段可能代表羚牛染色体着丝点的卫星DNA单体。     

Polymorphic microsatellites developed by cross-species amplifications in common pheasant breeds

Genetic variability was analysed in two common breeds of pheasant (Phasianus colchicus L. 1758) by means of cross-species amplifications of microsatellite loci: 154 chicken, Gallus gallus and 32 turkey, Meleagris gallopavo, primers were tested for amplification of pheasant DNA. Thirty-six primers (25 specific for chicken and 11 for turkey) amplified pheasant DNA. Fifteen markers yielded specific products and were tested for polymorphism. Eight of them (55%) were polymorphic, with an average polymorphism of two alleles per locus. Specific polymerase chain reaction (PCR) products were sequenced; repeats were found in 11 of the 15 markers, although only two loci showed the same repeat and could be homologous to chicken ones.     

DNA sequence organisation in avian genomes

By means of renaturation kinetics of DNA of the three avian species Cairina domestica, Gallus domesticus and Columba livia domestica the following major DNA repetition classes were observed: a very fast reannealing fraction comprising about 15% of the DNA, a fast or intermediate reannealing fraction that makes up 10%, and a slow reannealing fraction of about 70%, which apparently renatures with single copy properties. — Comparing the reassociation behaviour of short (0.3 kb) and long (>2 kb) DNA fragments of duck and chicken it becomes apparent that only 12% (duck) and 28% (chicken) of the single copy DNA are interspersed with repetitive elements on 2 to 3 kb long fragments. The lengths of the repetitive sequences were estimated by optical hyperchromicity measurements, by agarose A-50 chromatography of S₁ nuclease resistant duplexes and by electron microscopic measurements of the S₁ nuclease resistant duplexes. It was found that in the case of the chicken DNA the single copy sequences alternating with middle repetitive ones are at least 2.3 kb long; the interspersed moderate repeats have a length average of at least 1.5 kb. The sequence length of the moderate repeats in duck DNA is smaller. The results show that the duck and the chicken genomes do not follow the short period interspersion pattern of genome organisation, characteristic of the eucaryotic organisms studied so far.     

Cell killing by spleen necrosis virus is correlated with a transient accumulation of spleen necrosis virus DNA.

Spleen necrosis virus productively infects avian and rat cells. The average number of molecules of unintegrated and integrated viral DNA in cells at different times after infection was determined by hybridization and transfection assays. Shortly after infection, there was a transient accumulation of an average of about 150 to 200 molecules of unintegrated linear spleen necrosis virus DNA per chicken, turkey, or pheasant cell. No such accumulation was seen in infected rat cells. Soon after infection there was in chicken cells, but not inturkey, pheasant, or rat cells, also a transient integration of an average of 35 copies of viral DNA per cell. By 10 days after infection, the majority of this integrated viral DNA was lost from the population of infected chicken cells. At the same time, the majority of the unintegrated viral DNA was also lost from infected chicken, turkey, and pheasant cells. The transient cytopathic effect seen in these infected cells also occurred at this time. Late after infection about five copies of apparently nondefective spleen necrosis proviruses were stably integrated at multiple sites in chicken, turkey, pheasant, and rat DNA. These results demonstrate a correlation between the transient accumulation of large numbers of spleen necrosis virus DNA molecules and the transient occurrence of cytopathic effects.     

秦岭羚牛高度重复序列DNA片段的分子克隆和序列分析

羚牛(Budorcas taxicolor)属偶蹄目(Artiodactyla)、牛科(Bovidae),为我国一类大型珍贵保护动物。我们从其基因组中克隆得到若干约800bp的BamHI高度重复序列并对部分克隆进行了序列测定,发现它们显示了很高的同源性。利用其中一个单元为探针,对限制酶消化后的羚牛基因组DNA作杂交分析,发现其杂交谱带不具有个体及亚种间特异性,说明该重复序列在羚牛基因组中具有保守的分布和排列。在牛科动物中,羚牛BamHI片段与绵羊属和山羊属的相关序列具有高度同源性,而与水牛和家牛序列差异较大。这些结果为羚牛与羊亚科物种亲源关系较近的分类学观点提供了分子生物学证据。有证据表明,这些片段可能代表羚牛染色体着丝点的卫星DNA单体。     

A chicken middle-repetitive DNA sequence which shares homology with mammalian ubiquitous repeats.

We have identified and sequenced two members of a chicken middle repetitive DNA sequence family. By reassociation kinetics, members of this family (termed CRl) are estimated to be present in 1500-7000 copies per chicken haploid genome. The first family member sequenced (CRlUla) is located approximately 2 kb upstream from the previously cloned chicken Ul RNA gene. The second CRl sequence (CRl)Va) is located approximately 12 kb downstream from the 3' end of the chicken ovalbumin gene. The region of homology between these two sequences extends over a region of approximately 160 base pairs. In each case, the 160 base pair region is flanked by imperfect, but homologous, short direct repeats 10-15 base pairs in length. When the CRl sequences are compared with mammalian ubiquitous interspersed repetitive DNA sequences (human Alu and Mouse Bl families), several regions of extensive homology are evident. In addition, the short nucleotide sequence CAGCCTGG which is completely conserved in ubiquitous repetitive sequence families from several mammalian species is also conserved at a homologous position in the chicken sequences. These data imply that at least certain aspects of the sequence and structure of these interspersed repeats must predate the avian-mammalian divergence. It seems that the CRl family may possibly represent an avian counterpart of the mammalian ubiquitous repeats.     

Homology between avian oncornavirus RNAs and DNA from several avian species.

3H-labeled 35S RNA from avian myeloblastosis virus (AMV), Rous associated virus (RAV)-0, RAV-60, RAV-61, RAV-2, or B-77(w) was hybridized with an excess of cellular DNA from different avian species, i.e., normal or leukemic chickens, normal pheasants, turkeys, Japanese quails, or ducks. Approximately two to three copies of endogenous viral DNA were estimated to be present per diploid of normal chicken cell genome. In leukemic chicken myeloblasts induced by AMV, the number of viral sequences appeared to have doubled. The hybrids formed between viral RNA and DNA from leukemic chicken cells melted with a Tm 1 to 6 C higher than that of hybrids formed between viral RNA and normal chicken cell DNA. All of the viral RNAs tested, except RAV-61, hybridized the most with DNA from AMV-infected chicken cells, followed by DNA from normal chicken cells, and then pheasant DNA. RAV-61 RNA hybridized maximally (39%) with pheasant DNA, followed by DNA from leukemic (34%), and then normal (29%) chicken cells. All viral RNAs tested hybridized little with Japanese quail DNA (2 to 5%), turkey DNA (2 to 4%), or duck DNA (1%). DNA from normal chicken cells contained only 60 to 70% of the RAV-60 genetic information, and normal pheasant cells lacked some RAV-61 DNA sequences. RAV-60 and RAV-61 genomes were more homologous to the RAV-0 genome than to the genome of RAV-2, AMV, or B-77(s). RAV-60 and RAV-61 appear to be recombinants between endogenous and exogenous viruses.     

Isolation and characterization of a major tandem repeat family from the human X chromosome.

We report the identification and characterization of a family of repeated restriction fragments whose molecular organization is apparently specific to the human X chromosome. This fragment, identified as an ethidium bromide-staining 2.0 kilobase (kb) band in BamHI-digested DNA from a Chinese hamster-human somatic cell hybrid containing a human X chromosome, has been cloned into pBR325 and characterized. The 2.0 kb repeated family has been assigned to the Xp11 leads to Xq12 region on the X by Southern blot analysis of somatic cell hybrids and is predominantly arranged in tandem clusters of up to seven 2.0 kb monomers. Homologous DNA sequences, not organized as 2.0 kb BamHI fragments, are found elsewhere on the X chromosome and on at least some autosomes, but are not found on the Y chromosome. From a dosing experiment using various amounts of the cloned repeat, we estimate that there are 5,000-7,500 copies of the 2.0 kb BamHI repeat per haploid genome. Since the vast majority, if not all, of these are confined to the X chromosome, this repeated DNA family must account for 5-10% of all X chromosome DNA and must constitute the major sequence component of the pericentromeric region of the X.