Evolution of a B2 tagged sequence from a long-range repeat family in the genus Mus

A long-range repeat family of more than 50 kb repeat size is clustered in Chromosomes (Chr) 1 of Mus musculus and M. spretus. In M. musculus this long-range repeat family shows considerable variation of copy-number frequency and contains coding regions for at least two genes. In an intron of a gene, which is part of the repeat, a B2 small interspersed repetitive element (SINE) is inserted at identical positions. The B2 element is present in all copies of the long-range repeat family; it was presumably a component of the ancestral single-copy precursor sequence that gave rise by amplification to the repeat family. Copies of the long-range repeat family vary with respect to the number of TAAA tandem repeats in the A-rich 3 end region of the B2 element. As inferred from polymerase chain reaction (PCR) data, presence and frequency of repeat number variants in the (TAAA)_n block are strain and species specific. The B2 element and its flanking regions were sequenced from two copies of the long-range repeat family. Sequence divergence between the two copies (only non-CG base substitutions and deletions/insertions) was determined to be 2.6%. Based on the drift rate in human Alu elements and a correction for the higher drift rates in rodents, and estimate for the divergence time of 1.7 million years was calculated. Since the long-range repeat family is present in M. musculus and M. spretus, it must have evolved by amplification before the separation of the two species about 1–4 million years ago.     

A novel transposon-like repeat interrupted by an LTR element occurs in a cluster of B1 repeats in the mouse c-mos locus.

The mouse genomic locus containing the oncogene c-mos was analyzed for repetitive DNA sequences. We found a single B1 repeat 10 kb upstream and three B1 repeats 0.6 kb, 2.7 kb, and 5.4 kb, respectively, downstream from c-mos. The B1 repeat closest to c-mos contains an internal 7-bp duplication and a 18-bp insertion. Localized between the last two B1 repeats is a copy of a novel mouse repeat. Sequence comparison of three copies of this novel repeat family shows that they a) contain a conserved BglII site, b) are approximately 420 bp long, c) possess internal 50-bp polypurine tracts, and d) have structural characteristics of transposable elements. They are present in about 1500 copies per haploid genome in the mouse, but are not detectable in DNA of other mammals. The BglII repeat downstream from c-mos is interrupted by a single 632-bp LTR element. We estimate that approximately 1200 copies of this element are present per haploid genome in BALB/c mice. It shares sequence homology in the R-U5 region with an LTR element found in 129/J mice.     

Chromosome-specific alpha satellite DNA from human chromosome 1: hierarchical structure and genomic organization of a polymorphic domain spanning several hundred kilobase pairs of centromeric DNA

The human alpha satellite repetitive DNA family is organized as distinct chromosome-specific subsets localized to the centromeric region of each chromosome. Here, we report he isolation and characterization of cloned repeat units which define a hierarchical subset of alpha satellite on human chromosome 1. This subset is characterized by a 1.9-kb higher-order repeat unit which consists of 11 tandem approximately 171-bp alpha satellite monomer repeat units. The higher-order repeat unit is itself tandemly repeated, present in at least 100 copies at the centromeric region of chromosome 1. Using pulsed-field gel electrophoresis we estimate the total array length of these tandem sequences at the centromere of chromosome 1 to be several hundred kilobase pairs. Under conditions of high stringency, the higher-order repeat probe hybridizes specifically to chromosome 1 and can be used to detect several associated restriction fragment length DNA polymorphisms. As such, this probe may be useful for molecular and genetic analyses of the centromeric region of human chromosome 1.     

The chAB4 and NF1-related long-range multisequence DNA families are contiguous in the centromeric heterochromatin of several human chromosomes

We have investigated the large-scale organization of the human chAB4-related long-range multisequence family, a low copy-number repetitive DNA located in the pericentromeric heterochromatin of several human chromosomes. Analysis of genomic clones revealed large-scale (~100 kb or more) sequence conservation in the region flanking the prototype chAB4 element. We demonstrated that this low copy-number family is connected to another long-range repeat, the NF1-related (ΨNF1) multisequence. The two DNA types are joined by an ~2 kb-long tandem repeat of a 48-bp satellite. Although the chAB4- and NF1-like sequences were known to have essentially the same chromosomal localization, their close association is reported here for the first time. It indicates that they are not two independent long-range DNA families, but are parts of a single element spanning ~200 kb or more. This view is consistent both with their similar chromosomal localizations and the high levels of sequence conservation among copies found on different chromosomes. We suggest that the master copy of the linked chAB4–ΨNF1 DNA segment appeared first on the ancestor of human chromosome 17.     

Banded krait minor-satellite (Bkm)-associated Y chromosome-specific repetitive DNA in mouse.

The mouse Y chromosome remains highly condensed in all somatic tissues but decondenses extensively in testis. We have isolated a mouse Y chromosome-specific repeat M34 (11.5 kb) and shown that this is distributed along the Y chromosome except the sex-determining region (the Y short arm) in which GATA repeats are predominantly concentrated. It has 32 copies of GATA repeats in a 2.7 kb fragment. About 200-300 copies of M34 on the Y chromosome are interspersed among other sequences. A 1.2 kb fragment (p3) of M34, containing GATA repeats, also has scaffold attachment region (SAR) motifs which bind to nuclear matrices. A strong affinity of histone H1 to SAR motifs is implicated in maintaining the condensed state of the Y chromosome in somatic tissues. The probable significance of molecular organization of the Y chromosome is discussed.     

Characterization of Ngef, a novel member of the Dbl family of genes expressed predominantly in the caudate nucleus

We have identified Ngef as a novel member of the family of Dbl genes. Many members of this family have been shown to function as guanine nucleotide exchange factors for the Rho-type GTPases. Ngef is predominantly expressed in brain, with the strongest signal in the caudate nucleus, a region associated with the control of movement. Ngef contains a translated trinucleotide repeat, a polyglutamic acid stretch interrupted by a glycine. We have localized the Ngef gene to mouse chromosome 1 and the human homologue of Ngef to human chromosome 2q37. We have shown in preliminary experiments that Ngef has transforming potential in cell culture and is able to induce tumors in nude mice.     

Genetic mapping and assignment of a long-range repeat cluster to band D of chromosome 1 in Mus musculus and M. spretus.

A cluster (D1Lub1) of a long-range repeat family was mapped to the proximal part of the Giemsa-negative band D in Chromosome 1 of Mus musculus and M. spretus by in situ hybridization with cloned probes of the long-range repeat family. By making use of restriction fragment length polymorphisms in DNAs from interspecific backcross mice, the cluster could be mapped to a position 5.3 +/- 2.1 cM distal to the Inha locus and the same distance proximal to the Bcl-2 locus. D1Lub1 was inseparable in 114 meiotic events from Acrg, Sag, and Akp-3. Taken together, the data may serve as a reference for coordinating the genetic and cytogenetic maps of Mus Chromosome 1. High-copy-number variants of the cluster, which appear cytogenetically as homogeneously staining regions at the same chromosome location, presumably arose by amplification of the long-range repeat family in situ.     

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.     

The FSHD-Associated Repeat, D4Z4, Is a Member of a Dispersed Family of Homeobox-Containing Repeats, Subsets of Which Are Clustered on the Short Arms of the Acrocentric Chromosomes

Facioscapulohumeral muscular dystrophy (FSHD) is an autosomal dominant neuromuscular disorder that maps to human chromosome 4q35. FSHD is tightly linked to a polymorphic 3.3-kb tandem repeat locus, D4Z4. D4Z4 is a complex repeat: it contains a novel homeobox sequence and two other repetitive sequence motifs. In most sporadic FSHD cases, a specific DNA rearrangement, deletion of copies of the repeat at D4Z4, is associated with development of the disease. However, no expressed sequences from D4Z4 have been identified. We have previously shown that there are other loci similar to D4Z4 within the genome. In this paper we describe the isolation of two YAC clones that map to chromosome 14 and that contain multiple copies of a D4Z4-like repeat. Isolation of cDNA clones that map to the acrocentric chromosomes and Southern blot analysis of somatic cell hybrids show that there are similar loci on all of the acrocentric chromosomes. D4Z4 is a member of a complex repeat family, and PCR analysis of somatic cell hybrids shows an organization into distinct subfamilies. The implications of this work in relation to the molecular mechanism of FSHD pathogenesis is discussed. We propose the name 3.3-kb repeat for this family of repetitive sequence elements.     

Evaluation of the Stag3 gene and the synaptonemal complex in a rat model (as/as) for male infertility

Affected males (as/as) from the mutant TT rat strain are sterile due to spermatogenesis impairment with meiotic arrest at the pachytene stage. The as locus is on rat chromosome 12, in a region that shows conserved synteny to cM 74-94 on mouse chromosome 5. Stag3, a new member of the stromalin protein family, is expressed specifically in testis and associates to the synaptonemal complex. Mouse Stag3 gene has been assigned to cM 78 on chromosome 5. In this study, we have characterized the rat Stag3 gene and examined it as a candidate for male infertility in as/as rats. The rat Stag3 cDNA is 4181 nucleotides long, contains a highly polymorphic hexanucleotide repeat in the coding region, and encodes a 1256 amino acid protein with 93 and 77% sequence identity to mouse and human Stag3 proteins, respectively. No mutations or differences in size or abundance of Stag3 mRNA were detected between as/as and control rats, suggesting that Stag3 is not responsible for the aspermic phenotype. In addition, immunohistochemistry with antibodies against SCP1 and SPC3 proteins suggest that the synaptonemal complex structures are not primarily affected in these rats.     

A new family of widely propagated MB1-repeats in the human genome

A new family of repeats--i.e. MB1 repeats family--the number of copies of which per a human genome constitutes a few hundreds of thousands of copies has been revealed in a human gemone by computer analysis of a noncanonical similarity of nucleic acid sequences. The numbers of that family of repeats have also been revealed in the genomes of mouse and rat, they have been identified as mirror--reflected copies--in purines and pyrimidines--of B1 repeats in the genome of mouse and the Alu repeats in the human genome. The MB1 repeats tend to remain most similar at a length of 70 b.p. They are not flanked by short repeats, neither contain poly(A) region at the 3' end, by which they differ from the repeats of the SINE family. It has been assumed that the member of the Alu repeats family and the MB1 repeats family can form a so called H-form of DNA. The mirror-reflected repeat family could have been formed by replication of parallel DNA strands.     

Structure, organization, and sequence of alpha satellite DNA from human chromosome 17: evidence for evolution by unequal crossing-over and an ancestral pentamer repeat shared with the human X chromosome.

The centromeric regions of all human chromosomes are characterized by distinct subsets of a diverse tandemly repeated DNA family, alpha satellite. On human chromosome 17, the predominant form of alpha satellite is a 2.7-kilobase-pair higher-order repeat unit consisting of 16 alphoid monomers. We present the complete nucleotide sequence of the 16-monomer repeat, which is present in 500 to 1,000 copies per chromosome 17, as well as that of a less abundant 15-monomer repeat, also from chromosome 17. These repeat units were approximately 98% identical in sequence, differing by the exclusion of precisely 1 monomer from the 15-monomer repeat. Homologous unequal crossing-over is suggested as a probable mechanism by which the different repeat lengths on chromosome 17 were generated, and the putative site of such a recombination event is identified. The monomer organization of the chromosome 17 higher-order repeat unit is based, in part, on tandemly repeated pentamers. A similar pentameric suborganization has been previously demonstrated for alpha satellite of the human X chromosome. Despite the organizational similarities, substantial sequence divergence distinguishes these subsets. Hybridization experiments indicate that the chromosome 17 and X subsets are more similar to each other than to the subsets found on several other human chromosomes. We suggest that the chromosome 17 and X alpha satellite subsets may be related components of a larger alphoid subfamily which have evolved from a common ancestral repeat into the contemporary chromosome-specific subsets.     

Wdr12, a mouse gene encoding a novel WD-Repeat Protein with a notchless-like amino-terminal domain

The WD-repeat protein family consists of a large group of structurally related yet functionally diverse proteins found predominantly in eukaryotic cells. These factors contain several (4-16) copies of a recognizable amino-acid sequence motif (the WD unit) thought to be organized into a "propeller-like" structure involved in protein-protein regulatory interactions. Here, we report the cloning of a mouse cDNA, referred to as Wdr12, which encodes a novel WD-repeat protein of 423 amino acids. The WDR12 protein was predicted to contain seven WD units and a nuclear localization signal located within a protruding peptide between the third and fourth WD domains. The amino-terminal region shows similarity to that of the Notchless WD repeat protein. Sequence comparisons revealed WDR12 orthologs in various eukaryotic species. Wdr12 seems to correspond to a single-copy gene in the mouse genome, located within the C1-C2 bands of chromosome 1. These data, together with the results of Wdr12 gene expression studies and evidence of in vitro binding of WDR12 to the cytoplasmic domain of Notch1, led us to postulate a function for the WDR12 protein in the modulation of Notch signaling activity.     

Co-amplification of L1 line elements with localised low copy repeats in Giemsa dark bands: implications for genome organisation.

A repeat sequence island, located at the A3 Giemsa dark band on the mouse X chromosome and consisting of 50 copies of a localised long complex repeat unit (LCRU), features an unusually high concentration of L1 LINE repeat sequences juxtaposed and inserted within the LCRU. Sequence analysis of three independent genomic clones containing L1 LINE elements juxtaposed with the LCRU demonstrates a common junction sequence at the L1/LCRU boundary, suggesting that the high concentration of L1 LINE sequences in the repeat sequence island has arisen by association of an L1 element with an LCRU followed by amplification. The LCRU target site at this common junction sequence bears no resemblance to the target site of an L1 element inserted within one LCRU, indicating there is no specific preferential target site for L1 integration. We propose that co-amplification of L1 LINE elements with localised low copy repeat families throughout the genome could have a major effect on the chromosomal distribution of L1 LINE elements.     

The gene encoding TBC1D1 with homology to the tre-2/USP6 oncogene, BUB2, and cdc16 maps to mouse chromosome 5 and human chromosome 4

TBC1D1 is the founding member of a family of related proteins with homology to tre-2/UPS6, BUB2, and cdc16 and containing the tbc box motif of 180-220 amino acids. This protein family is thought to have a role in differentiation and in regulating cell growth. We set out to map the TBC1D1 gene in mouse and human. Segregation analysis of a TBC1D1 RFLP in two independent mouse RI (recombinant inbred) lines reveals that mouse Tbc1d1 is closely linked to Pgm1 on chromosome 5. The human TBC1D1 gene was assigned to human chromosome 4p15.1-->4q21 using Southern blot analyses of genomic DNAs from rodent-human somatic cell lines. A human-specific genomic fragment was observed in the somatic cell lines containing human chromosome 4 or the 4p15.1-->4q21 region of the chromosome. TBC1D1 maps to the region containing the ortholog of mouse Pgm1 adding another locus to this long region of conserved synteny between mouse and man.     

Isolation and characterization of an alphoid centromeric repeat family from the human Y chromosome

A collection of human Y-derived cosmid clones was screened with a plasmid insert containing a member of the human X chromosome alphoid repeat family, DXZ1. Two positive cosmids were isolated and the repeats they contained were investigated by Southern blotting, in situ hybridization and sequence analysis. On hybridization to human genomic DNAs, the expected cross-hybridization characteristic of all alphoid sequences was seen and, in addition, a 5500 base EcoRI fragment was found to be characteristic of a Y-specific alphoid repeat. Dosage experiments demonstrated that there are about 100 copies of this 5500 base EcoRI alphoid fragment on the Y chromosome. Studies utilizing DNA from human-mouse hybrids containing only portions of the Y chromosome and in situ hybridizations to chromosome spreads demonstrated the Y centromeric localization of the 5500 base repeat. Cross-hybridization to autosomes 13, 14 and 15 was also seen; however, these chromosomes lacked detectable copies of the 5500 base EcoRI repeat sequence arrangement. Sequence analysis of portions of the Y repeat and portions of the DXZ1 repeat demonstrated about 70% homology to each other and of each to the human consensus alphoid sequence. The 5500 base EcoRI fragment was not seen in gorilla, orangutan or chimpanzee male DNA.     

Evolution of a long-range repeat family in Chromosome 1 of the genus Mus

Copy numbers and variation of a clustered long-range repeat family on Chromosome (Chr) 1 have been studied in different species of the genus Mus. The repeat sequence was present in all, as inferred from cross-hybridization with probes derived from the Mus musculus repeat family. Copy numbers determined by dot blot hybridization were very low, from three to six per haploid genome in M. caroli, M. cervicolor, and M. cookii. These species form one branch of the phylogenetic tree in the genus Mus. In the other group of phylogenetically related species—M. spicilegus, M. spretus, M. musculus and M. macedonicus—copy numbers ranged from 6 to 1810 per haploid genome. The repeat cluster is cytogenetically visible as a fine C-band in M. macedonicus and as a C-band positive homogeneously staining region (HSR) in several populations of M. m. domesticus and M. m. musculus. When cytogenetically visible, the clusters contained from 179 to 1810 repeats. Intragenomic restriction fragment length polymorphisms (RFLPs), which reflect sequence variation among different copies of the long-range repeat family, increased with higher copy numbers. The high similarity of the RFLP pattern among genomes with C-band positive regions in Chr 1 of M. m. musculus, M. m. domesticus, and M. macedonicus points to a close evolutionary relationship of their Chr 1 repeat families.     

Identification of VCR, a repeated sequence associated with a locus encoding a hemagglutinin in Vibrio cholerae O1.

We have determined the nucleotide sequence of a 6.3-kb BamHI fragment of the chromosome of Vibrio cholerae 569B that includes the sequence of the mannose-fucose-resistant hemagglutinin reported previously (V.L. Franzon, A. Barker, and P. A. Manning, Infect. Immun. 61:3032-3037, 1993). This region contains nine copies of a 124-bp direct repeat, here named VCR, of imperfect dyad symmetry, that are shown by Southern hybridization to occur at least 60 to 100 times in the V. cholerae O1 chromosome. Large-scale chromosomal mapping suggests that the repeats are confined to about 10% of the chromosome. Related sequences are also found in non-O1 V. cholerae but not in other members of the family Vibrionaceae. However, VCR is unrelated to other previously described repetitive sequences.     

Physical mapping of sequences homologous to an endogenous retrovirus LTR on human chromosome 19

The human genome contains multiple copies of sequences related to the HERV-K family of endogenous retroviruses, homologous to the B-type mouse mammary tumour virus. A DNA fragment closely resembling an HERV-K long tandem repeat (LTR) was detected in a library of hncDNA clones enriched for sequences from human chromosome 19. Sites showing homology to the sequence of this fragment have been identified on human chromosome 19 by hybridization to previously mapped chromosome 19 cosmids. Thus the distribution of LTR sequences on a specific human chromosome has been mapped for the first time. We estimate the total number of such sites on human chromosome 19 to be at least 110. Many of these sites are located in the vicinity of known genes. The precise localizations (to specific cosmids) of LTR-homologous sequences on chromosome 19 can serve as a reference source and will automatically provide further insight into LTR-gene relationships as new genes are mapped onto the chromosome.