Isolation of Y Chromosome-Specific Sequences from Silene Latifolia and Mapping of Male Sex-Determining Genes Using Representational Difference Analysis

The genomic subtraction method representational difference analysis (RDA) was used to identify male-specific restriction fragments in the dioecious plant Silene latifolia. Male-specific restriction fragments are linked to the male sex chromosome (the Y chromosome). Four RDA-derived male-specific restriction fragments were used to identify polymorphisms in a collection of X-ray-generated mutant plants with either hermaphroditic or asexual flowers. Some of the mutants have cytologically detectable deletions in the Y chromosome that were correlated with loss of male-specific restriction fragments. One RDA-derived probe detected a restriction fragment present in all mutants, indicating that each has retained Y chromosomal DNA. The other three probes detected genomic fragments that were linked in a region deleted in some hermaphroditic and some asexual mutants. Based on the mutant phenotypes and the correlation of cytologically visible deletions with loss of male-specific restriction fragments, these markers were assigned to positions on the Y chromosome close to the carpel suppression locus. This RDA mapping also revealed a Y-linked locus, not previously described, which is responsible for early stamen development.     

Y chromosome DNA polymorphisms in two African populations.

Y chromosome-specific DNA polymorphisms were detected using probe p49f after restriction with TaqI enzyme on samples coming from two African populations: Bantus and Pygmies. All the main TaqI alleles at five Y loci already found in Caucasians are also found in these two populations; 12 of the 16 Caucasian haplotypes were found in these two African populations, and two new haplotypes are Pygmy specific. A proposed phylogeny of the various haplotypes that was derived by using the parsimony criterion established that haplotypes XIII and XVIII, respectively the most frequent one and only one present in Pygmies, are probably ancestral.     

Rearrangements of highly polymorphic regions near telomeres of Saccharomyces cerevisiae.

We have examined the mitotic and meiotic properties of telomeric regions in various laboratory strains of yeast. Using a sequence (Y probe) derived from a cloned yeast telomere (J. Szostak and E. Blackburn, Cell 29:245-255, 1982), we found that various strains of Saccharomyces cerevisiae show extensive polymorphisms of restriction endonuclease fragment length. Some of the variation in the lengths of telomeric fragments appears to be under the control of a small number of genes. When DNA from various strains was digested with endonuclease KpnI, nearly all of the fragments homologous to the Y probe were found to be of different size. The pattern of fragments in different strains was extremely variable, with a greater degree of polymorphism than that observed for fragments containing the mobile TY1 element. Tetrad analysis of haploid meiotic segregants from diploids heterozygous for many different Y-homologous KpnI fragments revealed that most of them exhibited Mendelian (2:0) segregation. However, only a small proportion of these fragments displayed the obligate 2:2 parental segregation expected of simple allelic variants at the same chromosome end. From the segregations of these fragments, we concluded that some yeast telomeres lack a Y-homologous sequence and that the chromosome arms containing a Y-homologous sequence are different among various yeast strains. Regions near yeast telomeres frequently undergo rearrangement. Among eight tetrads from three different diploids, we have found three novel Y-homologous restriction fragments that appear to have arisen during meiosis. In all three cases, the appearance of a new fragment was accompanied by the loss of another band. In one of these cases, the rearrangement leading to a novel fragment arose in an isogenic diploid, in which both homologous chromosomes should have been identical. Among these same tetrads we also found examples of apparent mitotic gene conversions and mitotic recombination involving telemetric regions.     

DNA cloning and sequence analysis of chicken AFLP

Amplified fragment length polymorphisms (AFLP) have been shown to be useful for linkage mapping in chickens and other domestic animals. It is often desirable to convert AFLP bands to sequence-tagged site (STS) markers, in particular, so that AFLP-based linkage information can be integrated with recombinant DNA clone-based maps. Sixteen chicken AFLP bands were excised from gels, re-amplified, cloned and analysed. All inserts proved to be EcoRI-TaqI fragments, which suggests that unlabelled TaqI-TaqI AFLP fragments do not amplify well, and therefore do not significantly contaminate AFLP bands. For eight of the AFLP, the cloned fragment was used to probe blots of AFLP reaction fingerprints, confirming that the predominant DNA clone indeed contained the polymorphic fragment. Flanking regions of selected AFLP fragments were isolated using Vectorette cloning. The results obtained suggest that the these chicken AFLP most commonly arise from sequence polymorphism at or near the TaqI site.     

Linkage studies of polymorphic, repeated DNA sequences in centromeric regions of human chromosomes.

The DNA at human centromeric regions was characterized by using a repetitive sequence, 308, which localizes in situ exclusively to centromeres of all chromosomes. We previously noted that this sequence is enriched on chromosome 6 and has chromosome-specific organization on 6, 3, 7, 14, X, and Y. In addition to this basic organization, sequences homologous to 308 are polymorphic among normal individuals. The variants are transmitted in a Mendelian manner within a family. To determine the chromosome origin of the variants, we studied their linkage to markers of various chromosomes. Linkage analysis of one pedigree segregating two polymorphisms shows that the 2.6-kilobase (kb) BamHI and 2.6-kb TaqI fragments are linked to each other and to the HLA loci on chromosome 6. Data from another family shows that 2.8-kb TaqI, 4.0-kb TaqI, and 1.3-kb BamHI polymorphic fragments are linked and are probably near the Fy locus on chromosome 1. By dot blot analysis, we determined that the relative amount of these sequences in the genome is not measurably different between unrelated individuals. Thus, the polymorphisms represent changes in homologous 308 sequences on specific chromosomes and can be used as chromosome-specific markers. Linkage studies using polymorphisms of repeated sequences will be most useful within a kindred, especially from an inbred population, because polymorphic repeats of the same restriction size may be heterogeneous in origin.     

Associations between restriction fragment length polymorphisms detected with a probe for human 21-hydroxylase (21-OH) and two clinical forms of 21-OH deficiency

Summary DNAs from unrelated healthy individuals and unrelated individuals affected with 21-hydroxylase deficiency (congenital and late-onset adrenal hyperplasia) were digested with seven restriction enzymes and hybridized with a cDNA probe specific for human 21-hydroxylase genes. Associations were found between restriction fragments and the two forms of the disease: (i) The late onset form is associated with a double dose of a 14 kb fragment generated by Eco RI and with a triple dose of a 3.2 kb fragment generated by TaqI in patients with HLA B14 haplotypes; (ii) The classical congenital form is negatively associated with the 14 kb fragment and with a 3.7 kb fragment generated by TaqI in patients with HLA Bw47 haplotypes. A 3.2 kb TaqI fragment is negatively associated with the HLA B8 haplotypes. The other five enzymes tested give no polymorphisms or polymorphisms without correlation with the two forms of the disease.     

Ethnic-specific allelic variation within the D1Z2 locus

DNAs from 122 individuals representing 5 ethnic groups (Black, Chinese, Japanese, Caucasian and Melanesian) were analyzed for restriction fragment length polymorphisms (RFLPs) with a hypervariable repeated sequence located uniquely on chromosome 1 (hMF No.1; is a component of the D1Z2 locus). When human genomic DNA is digested with a variety of enzymes (TaqI, EcoRI, SinI, PstI, HaeIII) the hMF No.1 probe reveals multiple RFLPs. Ethnic group differences were found in the frequencies of specific EcoRI bands. The most striking ethnic group variation was the presence of a unique fragment amongst the Japanese.     

A search for restriction fragment length polymorphism on the human Y chromosome

Summary A systematic search for restriction fragment length polymorphisms (RFLPs) on the human Y chromosome was performed. DNA samples from 16–34 individuals were screened with five restriction enzymes and 12 Y-chromosomal probes, 3 of which detect lowly repetitive sequences and 9 of which are apparently single copy in genomic DNA. None of the single-copy probes revealed any variation. The repetitive sequence probe p21A1 (DYZ?) revealed a TaqI RFLP with q = 0.05. The frequency of fixed point mutations in Y-chromosomal DNA outside the pseudoautosomal region is probably less than 1 in 18000 bp.     

Strategies for detecting restriction polymorphisms of Y chromosome sequences

Six cloned genomic sequences from the human Y chromosome were used for the detection of restriction polymorphisms in a panel of unrelated DNAs. Three sorts of strategies for the detection of polymorphisms were used: (a) mixing individual DNAs restricted by several endonucleases, (b) separated restrictions of the DNA library members with six different restriction enzymes, (c) individual DNA digestion with TaqI and MsqI. Results show that restriction polymorphisms are detected only rarely; this result can be explained by exemption of most of the Y chromosome from meiotic pairing and exchange.     

Isolation and characterization of Y chromosome DNA probes.

A sorted, cloned Y chromosome phage library was screened for unique Y chromosome sequences. Of the thousands of plaques screened, 13 did not hybridize to radiolabeled 46,XX total chromosomal DNA. Three plaques were characterized further. Clone Y1 hybridized to multiple restriction enzyme fragments in both male and female DNA with more intense bands in male DNA. Clone Y2, also found in female and male DNA, is probably located in the pseudosutosomal region because extra copies of either the X or Y chromosomes increased Y2 restriction enzyme fragment intensity in total cellular DNA. Clone Y5 was male specific in three of four restriction enzyme digests although in the fourth a light hybridizing band was observed in both male and female DNA. Clone Y5 was sublocalized to band Yq 11.22 by hybridization to a panel of cellular DNA from patients with Y chromosome rearrangements. Clone Y5 can be used to test for retention of the proximally long arm Y suggested to cause gonadal cancer in carrier females. The long series of GA repeats in Y5, anticipated to be polymorphic, may provide a sensitive means to follow Y chromosome variation in human populations.     

Restriction fragment length polymorphism of DQB and DRB class II genes of the ovine major histocompatibility complex

The ovine major histocompatibility complex (MhcOvar) class II region was investigated by Southern blot hybridizations using ovine probes specific for the second exons of Ovar-DRB and Ovar-DQB genes. Multiple bands were revealed when genomic DNA was digested with each of five restriction enzymes (Bam HI, Eco RI, Hin dIII, PvuII and TaqI), and successively hybridized with the two radiolabeled ovine probes. Restriction fragment length polymorphisms (RFLPs) were analysed in 89 sheep originating from six inbred families and the inheritance of the fragment patterns was determined. Forty-one fragments were recorded with the DQB probe; 32 were detected with the DRB probe. They constituted 9 DQB and 10 DRB allelic patterns. Twelve DQB-DRB haplotypes were resolved in this study.     

Mouse ferritin H multigene family is polymorphic and contains a single multiallelic functional gene located on chromosome 19.

Multiple ferritin H subunit sequences are present in the genome of higher vertebrates, but it is not yet known with certainty if more than one is expressed. In this paper, we provide evidence that there is only one functional ferritin H gene in the mouse. We screened a mouse genomic library using a mouse ferritin H cDNA as a probe and characterized five clones. These genomic clones proved to contain three pseudogenes and two allelic forms of a unique functional gene. These two alleles differed by only two point mutations in the promoter and three in the first intron and by a 31-bp insertion in the first intron. They were equally expressed when transiently transfected in HeLa cells. These five genomic clones account for all the bands observed on a Southern blot of mouse genomic DNA hybridized with a ferritin H cDNA, and these bands present a restriction fragment length polymorphism between various representatives of the genus Mus. Using a DNA panel prepared from the backcross progeny (C57BL/6 X Mus spretus)F1 X C57BL/6, we localized the functional ferritin H gene (Fth) in region B of mouse chromosome 19 and established cen-Ly-1-Fth-Pax-2 as the most likely gene order, thus defining a conserved syntenic fragment with human chromosome 11q.     

Genomic clone OS-2 (D10S20) detects different restriction fragment length polymorphisms in Caucasians and Orientals for both HindIII and TaqI.

Restriction fragment length polymorphisms (RFLPs) for the anonymous DNA probe OS-2 were studied in a Caucasian population. In a sample of 15 two- and three-generation families and 31 unrelated individuals, the restriction endonuclease TaqI revealed an RFLP not seen in a Japanese sample. A similar situation has been observed for this probe with the restriction endonuclease HindIII.     

Molecular differentiation of the homomorphic sex chromosomes inMegaselia scalaris (Diptera) detected by random DNA probes

Randomly cloned DNA fragments and a poly-(GATA) containing sequence were used as probes to identify sex chromosomal inheritance and to detect differences at the molecular level between the homomorphic X and Y in the phorid fly,Megaselia scalaris. Restriction fragment length differences between males and females and between two laboratory stocks of different geographic origin were used to differentiate between sex chromosomal and autosomal origin of the respective fragments. Five random probes detected X and Y chromosomal DNA loci and two others recognized autosomal DNA loci. One random probe and the poly(GATA) probe hybridized with both sex chromosomal and autosomal restriction fragments. Most of the Y chromosomal restriction fragments were conserved in length between the two stocks while most of the X chromosomal and autosomal fragments showed length polymorphism. It was concluded, therefore, that the Y chromosome contains a conserved segment in which crossover is suppressed and restriction site differences have accumulated relative to the X. These chromosomes, therefore, conform to a theoretically expected early stage of sex chromosome evolution.     

Multiple, independent restriction site polymorphisms in human DNA detected with a cDNA probe to argininosuccinate synthetase (AS).

We have used a cDNA clone of the human urea cycle enzyme argininosuccinate synthetase (AS) to screen for restriction fragment length polymorphisms (RFLPs) using a large panel of restriction enzymes. The probe, pAS-1, detects from 15 to 27 human DNA fragments by Southern gel analysis. In addition to the structural locus on chromosome 9, AS-like sequences are found on at least 10 human chromosomes, including the X and Y. This large number of dispersed pseudogenes accounts for the multiplicity of hybridizing fragments detected with pAS-1. Of 37 restriction enzymes tested, 18 produced excellent digest patterns; of these 18 enzymes, three revealed high-frequency, independent RFLPs, testing a minimum of 16 individuals with each enzyme. The enzymes producing high-frequency polymorphisms are Hind III (allele frequencies .30 and .70), Hind II (.13 and .87), and Bam HI (.56 and .44). Most of the polymorphic alleles are found in Caucasians, American blacks, and Orientals. The RFLP detected with Hind III maps to chromosome 9 (9q11-q22), although not to the structural locus; the others are autosomal but otherwise unassigned. Two additional six-base enzymes and a four-base enzyme, Msp I, revealed further individual variation, but these variants have not been shown to segregate in families yet. We observed approximately 0.8% high-frequency RFLPs per cut site per enzyme or a minimum of .05% heterozygosity per nucleotide. pAS-1 should prove useful in assigning high-frequency RFLPs to several human chromosomes.     

Carrier detection in haemophilia B using two further intragenic restriction fragment length polymorphisms.

A normal human population has been screened for the existence of further restriction fragment length polymorphisms (RFLPs) in the clotting factor IX gene in addition to the TaqI polymorphism already characterised (1,2). Two polymorphic loci were found, both within 6 Kb of the TaqI polymorphism within the body of the factor IX gene. One of the polymorphisms has been shown to be due to either the presence or absence of a particular recognition site for the restriction enzyme XmnI. The other, visualised as a difference in fragment pattern produced by digestion with either HinfI or DdeI, has two allelic forms differing by a 50 bp element of inserted DNA. Sequence analysis has shown the inserted element to be in a region of Z type DNA sequence, the insertion representing a duplication of flanking sequence on either side. The two polymorphisms are inherited in simple Mendelian fashion and have both been used to diagnose haemophilia B carrier status. It is estimated that the combined use of these polymorphisms in the factor IX gene, despite linkage disequilibrium between the 3 polymorphic loci, should enable carrier status to be determined in approximately 66% of all haemophilia B families.     

Alphoid DNA polymorphisms for chromosome 21 can be distinguished from those of chromosome 13 using probes homologous to both.

Although alphoid DNA sequences shared among acrocentric chromosomes have been identified, no human chromosome 21-specific sequence has been isolated from the centromeric region. To identify alphoid DNA restriction fragment length polymorphisms (RFLPs) specific for chromosome 21, we hybridized human genomic DNA with alphoid DNA probes [L1.26; aRI(680),21-208] shared by chromosomes 13 and 21. We detected RFLPs with restriction enzymes ECoRI, HaeIII, MboI,StuI, and TaqI. The segregation of these RFLPs was analyzed in the 40 CEPH families. Linkage analysis between these RFLPs and loci previously mapped to either chromosome 13 or 21 revealed RFLPs that appear to be specific to chromosome 21. These polymorphisms may be useful as genetic markers of the centromeric region of chromosome 21. Different alphoid loci within the centromeric region of chromosome 13 were identified.     

Novel restriction fragment length polymorphism of the growth hormone gene in inbred rats

A novel restriction fragment length polymorphism in inbred rats was detected by Southern blot analysis with rat growth hormone cDNA as a probe. Four alleles, characterized by PstI fragments of 1.2, 1.1, 0.9, and 0.7 kb, respectively, were detected in 27 strains examined. The same distribution of polymorphisms was observed on digestion of DNAs of these strains with three other enzymes, PvuII, HindIII, and BamHI. Moreover, the same differences in length of allelic restriction fragments were obtained with these restriction enzymes as with PstI. These findings suggested that the polymorphism was caused by insertion or deletion of variable DNA segments in the second intron of the growth hormone gene. Linkage analyses using backcross progeny provided no evidence for close linkage between the restriction fragment length polymorphism locus and 10 other loci examined.     

Organization and stability of endogenous xenotropic murine leukemia virus proviral DNA in mouse genomes.

In a series of blot hybridization experiments, using a xenotropic envelope probe and restriction enzymes known to cut xenotropic proviral DNA a single time (EcoRI) or not at all (HindIII), we have studied the organization and relationship of endogenous xenotropic env-related sequences in various mouse strains. Multiple copies (18 to 28) of xenotropic env-reactive fragments were found in all mouse DNAs after digestion with either HindIII or EcoRI, and the majority of fragments were of sizes compatible with their origin from full-length proviral DNA. Five HindIII and five EcoRI restriction fragments were common to all inbred mouse DNAs tested. In addition, each strain exhibited unique characteristic xenotropic env-reactive bands; these bands were remarkably stable during many years of inbreeding. The cleavage patterns characteristic of each strain were also useful for showing genealogical relatedness among the various inbred mice.