Isolation and initial characterization of a large repeat sequence element specific to mouse chromosome 8.

A clone containing 15.6 kb of mouse genomic DNA was specifically localized to murine chromosome 8 by fluorescence in situ hybridization. The major signal, mapping just below the centromeric heterochromatin, was much too intense for a single-copy probe. Two additional weak hybridization signals were detected in or near distal bands 8B3 and 8D. Six subclones spanning the entire 15.6-kb insert gave strong centromere proximal signals; however, none of these clones cross-hybridized with each other, suggesting that the repeat unit was quite large. Sequence data support this interpretation. An analysis of over 4 kb of sequence, including two subclones in their entirety, did not reveal any common sequence motif. Copy number reconstruction and Southern blotting experiments indicate that between 60 and 80 copies of the sequence (approximately 0.9-1.2 Mb in total) reside on each chromosome 8, most likely organized in a clustered but not tandemly duplicated fashion. Although the probe hybridizes to Mus spretus and Mus castaneus as well as to Mus musculus, it is not detectable in the rat, Chinese hamster, Armenian hamster, or human genomes.     

Isolation and Characterization of a Mouse Y Chromosomal Repetitive Sequence

The Y chromosome plays a dominant role in mammalian sex determination, and characterization of this chromosome is essential to understand the mechanism responsible for testicular differentiation. Male mouse genomic DNA fragments, cloned into pBR322, were screened for the presence of Bkm (a female snake satellite DNA)-related sequences, and we obtained a clone (AC11) having a DNA fragment from the mouse Y chromosome. In addition to a Bkm-related sequence, this fragment contained a Y chromosomal repetitive sequence. DNA isolated from the XX sex-reversed male genome produced a hybridization pattern indistinguishable to that obtained with normal female DNA, suggesting that the AC11 sequence is not contained within the Y chromosomal DNA present in the sex-reversed male genome. Based on the hybridization patterns against mouse Y chromosomal DNA, AC11 classified 16 inbred laboratory strains into two categories; those with the Mus musculus musculus type Y chromosome and those with the M.m. domesticus type Y chromosome. Three European subspecies of Mus musculus (M.m. brevirostris, M.m. poschiavinus and M.m. praetextus) possessed the M.m. domesticus type Y chromosome, whereas the Japanese mouse, M.m. molossinus, had the M.m. musculus type Y chromosome. The survey was also extended to six other species that belong to the genus Mus, of which M. spretus and M. hortulamus showed significant amounts of AC11-related sequences in their Y chromosomes. The male-specific accumulation of AC11-related sequences was not found in M. caroli, M. cookii, M. pahari or M. platythrix. This marked difference among Mus species indicates that the amplification of AC11-related sequences in the mouse Y chromosome was a recent evolutionary event.     

Rapid physical mapping of cloned DNA on banded mouse chromosomes by fluorescence in situ hybridization.

Physical mapping of DNA clones by nonisotopic in situ hybridization has greatly facilitated the human genome mapping effort. Here we combine a variety of in situ hybridization techniques that make the physical mapping of DNA clones to mouse chromosomes much easier. Hybridization of probes containing the mouse long interspersed repetitive element to metaphase chromosomes produces a Giemsa-like banding pattern which can be used to identify individual Mus musculus, Mus spretus, and Mus castaneus chromosomes. The DNA binding fluorophore, DAPI, gives quinacrine-like bands that can complement the hybridization banding data. Simultaneous hybridization of a differentially labeled clone of interest with the banding probe allows the assignment of a mouse clone to a specific cytogenetic band. These methods were validated by first mapping four known genes, Cpa, Ly-2, Cck, and Igh-6, on banded chromosomes. Twenty-seven additional clones, including twenty anonymous cosmids, were then mapped in a similar fashion. Known marker clones and fractional length measurements can also provide information about chromosome assignment and clone order without the necessity of recognizing banding patterns. Clones hybridizing to each murine chromosome have been identified, thus providing a panel of marker probes to assist in chromosome identification.     

Genome comparison in the genus Mus: a study with B1, MIF (mouse interspersed fragment), centromeric, and Y-chromosomal repetitive sequences

Using four repetitive sequences, we compared DNAs isolated from Mus caroli, M. cookii, M. hortulanus, M. musculus, M. pahari, M. saxicola, and M. spretus. Except for B1, these probes showed species-specific hybridization patterns. Mouse interspersed fragment (MIF) sequences were present in all species examined, but those defined by the 1.3-kb EcoR1 band were fewer in M. pahari and M. saxicola than in the other species. The Y-chromosomal probe showed male-specific accumulation only in M. hortulanus, M. musculus, and M. spretus, which are known to be closely related. The genetic difference between M. spretus and the other two species (M. hortulanus and M. musculus) was clearly demonstrated by a M. musuclus centromeric sequence that hybridized strongly to M. hortulanus and M. musculus DNA but was underrepresented in the genome of M. spretus. These results may suggest the usefulness of these repetitive sequences in the classification of Mus species that display only subtle morphological differences.     

Construction of a small Mus musculus repetitive DNA library: identification of a new satellite sequence in Mus musculus.

We report the construction of a small library of recombinant plasmids containing Mus musculus repetitive DNA inserts. The repetitive cloned fraction was derived from denatured genomic DNA by reassociation to a Cot value at which repetitive, but not unique, sequences have reannealed followed by exhaustive S1 nuclease treatment to degrade single stranded DNA. Initial characterizations of this library by colony filter hybridizations have led to the identification of a previously undetected M. musculus minor satellite as well as to clones containing M. musculus major satellite sequences. This new satellite is repeated 10-20 times less than the major satellite in the M. musculus genome. It has a repeat length of 130 nucleotides compared with the M. musculus major satellite with a repeat length of 234 nucleotides. Sequence analysis of the minor satellite has shown that it has a 29 base pair region with extensive homology to one of the major satellite repeating subunits. We also show by in situ hybridization that this minor satellite sequence is located at the centromeres and possibly the arms of at least half the M musculus chromosomes. Sequences related to the minor satellite have been found in the DNA of a related Mus species, Mus spretus, and may represent the major satellite of that species.     

Construction and utility of 10-kb libraries for efficient clone-gap closure for rice genome sequencing

Rice is an important crop and a model system for monocot genomics, and is a target for whole genome sequencing by the International Rice Genome Sequencing Project (IRGSP). The IRGSP is using a clone by clone approach to sequence rice based on minimum tiles of BAC or PAC clones. For chromosomes 10 and 3 we are using an integrated physical map based on two fingerprinted and end-sequenced BAC libraries to identifying a minimum tiling path of clones. In this study we constructed and tested two rice genomic libraries with an average insert size of 10 kb (10-kb library) to support the gap closure and finishing phases of the rice genome sequencing project. The HaeIII library contains 166,752 clones covering approximately 4.6x rice genome equivalents with an average insert size of 10.5 kb. The Sau3AI library contains 138,960 clones covering 4.2x genome equivalents with an average insert size of 11.6 kb. Both libraries were gridded in duplicate onto 11 high-density filters in a 5 x 5 pattern to facilitate screening by hybridization. The libraries contain an unbiased coverage of the rice genome with less than 5% contamination by clones containing organelle DNA or no insert. An efficient method was developed, consisting of pooled overgo hybridization, the selection of 10-kb gap spanning clones using end sequences, transposon sequencing and utilization of in silico draft sequence, to close relatively small gaps between sequenced BAC clones. Using this method we were able to close a majority of the gaps (up to approximately 50 kb) identified during the finishing phase of chromosome-10 sequencing. This method represents a useful way to close clone gaps and thus to complete the entire rice genome.     

Sequences and evolutionary analysis of mouse 5S rDNAs

We selectively amplified the spacer regions of genes for mouse 5S ribosomal RNA (rRNA), which are tandemly repeated, by the PCR method, using primers specific to the two ends of the coding region for 5S rRNA. Fragments of approximately 1.6 kb were amplified from DNA from the BALB/cCrSlc mouse (Mus musculus domesticus), the SM/J mouse (M. m. domesticus), the MOA mouse (M. m. musculus) and the SEG mouse (M. spretus). These fragments were cloned into an appropriate plasmid vector, and two clones representative of each of the four strains were sequenced. The sequences were GC rich (> 60%) and contained a high proportion of very simple repetitive motifs, such as (TG)n and (ATCC)n, which accounted for the intra- and intergenomic length heterogeneity. Excluding such polymorphic regions and neglecting small insertions or deletions, we estimated the sequence divergence between clones. Sequence divergence within a genome averaged 0.26%, and the divergence between individuals of the same subspecies, between subspecies, and between species was 0.44%, 0.62%, and 1.73%, respectively. The results indicate that the spacer region evolved rapidly but with a reduction in heterogeneity within each genome, as a result of certain, as yet unidentified, homogenization mechanisms. The results further suggest that the spacer regions of genes for 5S rRNA may provide good indicators for phylogenetic analysis of closely related species.      

Localization of the human thyroxine-binding globulin gene to the long arm of the X chromosome (Xq21-22).

Thyroxine-binding globulin (TBG) is the major thyroid-hormone transport protein in the plasma of most vertebrate species. A recombinant phage (lambda cTBG8) containing a cDNA insert of human TBG recently has been described. With the cDNA insert from lambda cTBG8 used as a radiolabeled probe, DNA from a series of somatic-cell hybrids containing deletions of the X chromosome was analyzed by means of blot hybridization. The results indicated that the TBG gene is located in the midportion of the long arm of the X chromosome between bands Xq11 and Xq23. The gene then was mapped to band region Xq21-22 by means of in situ hybridization to metaphase chromosomes. Sequences on the X chromosome that are homologous to the cDNA probe are contained within a single EcoRI restriction fragment of 12.5 kb in human DNA. On the basis of the intensity of the hybridization signal on Southern blots, it was determined that the human TBG cDNA probe used in the present study shares significant homology with hamster and mouse sequences. A single EcoRI restriction fragment was recognized in both hamster (8.0-kb) and mouse (5.1-kb) DNA.     

Mus Spretus Line-1 Sequences Detected in the Mus Musculus Inbred Strain C57bl/6j Using Line-1 DNA Probes

The inbred mouse strain, C57BL/6J, was derived from mice of the Mus musculus complex. C57BL/6J can be crossed in the laboratory with a closely related mouse species, M. spretus to produce fertile offspring; however there has been no previous evidence of gene flow between M. spretus and M. musculus in nature. Analysis of the repetitive sequence LINE-1, using both direct sequence analysis and genomic Southern blot hybridization to species-specific LINE-1 hybridization probes, demonstrates the presence of LINE-1 elements in C57BL/6J that were derived from the species M. spretus. These spretus-like LINE-1 elements in C57BL/6J reveal a cross to M. spretus somewhere in the history of C57BL/6J. It is unclear if the spretus-like LINE-1 elements are still embedded in flanking DNA derived from M. spretus or if they have transposed to new sites. The number of spretus-like elements detected suggests a maximum of 6.5% of the C57BL/6J genome may be derived from M. spretus.     

Human ornithine decarboxylase sequences map to chromosome regions 2pter----p23 and 7cen----qter but are not coamplified with the NMYC oncogene

Using a mouse cDNA probe for ornithine decarboxylase (ODC), we have identified and isolated an ODC cDNA clone from a lambda gt11 recombinant library prepared from human liver cell mRNA. The 2.0-kb insert of this clone hybridizes with several mouse genomic ODC DNA restriction fragments under conditions of low stringency, but reacts with only few human DNA fragments and a polyA+ RNA species of 2.2 kb under both nonstringent and stringent hybridization conditions. This suggests that, unlike the mouse genome, there are only few ODC genes in the human genome. The human ODC DNA fragments segregate with chromosome regions 2pter----p23 and 7cen----qter in mouse X human somatic cell hybrid clones containing normal, translocated, and deleted human chromosomes. Sequences of the short arm of chromosome 2 containing the NMYC oncogene at 2p23----p24 are often involved in DNA amplification in neuroblastomas and small-cell lung cancers. However, in at least three cases--one neuroblastoma cell line, one neuroblastoma tumor, and one lung carcinoma--the ODC sequences are not coamplified with the NMYC oncogene.     

Karyotypic evolution and organization of the highly repetitive DNA sequences in the Japanese shrew-moles, Dymecodon pilirostris and Urotrichus talpoides

The karyological relationship and organization of highly repetitive DNA sequences in Japanese shrew-moles were studied by zoo-blot hybridization and fluorescence in situ hybridization (FISH). When the genomic DNA of the eastern race of Urotrichus talpoides was digested with PstI, three fragments of highly repetitive DNA sequences, approximately 0.7, 0.9, and 1.4 kb in length, were observed as distinct bands. The results of FISH in the eastern race of U. talpoides using these three fragments separately as probes showed that the 0.7-kb PstI fragment was distributed in the centromeric regions of most chromosomes, and that the 0.9- and 1.4-kb fragments were predominantly located in the C-heterochromatin region of chromosome 13p. Although the western race of U. talpoides also had three PstI fragments, 0.9- and 1.4-kb PstI fragments were more ambiguous than those of the eastern race. The PstI- digested genomic DNA in Dymecodonpilirostris produced only a faint 0.9-kb band, and its signal patterns obtained by zoo-blot hybridization were clearly different from those of U. talpoides. The 0.7-kb fragment of U. talpoides hybridized strongly with the 0.9-kb fragment of D. pilirostris. In a FISH analysis, the 0.9-kb fragment of D. pilirostris hybridized with highly repetitive DNA in the centromeric regions of most chromosomes from both D. pilirostris and U. talpoides. Zoo-blot hybridization and FISH analyses suggest that the 0.9- and 1.4-kb PstI fragments were generated specifically in the genome of U. talpoides after the common ancestor differentiated into two extant shrew-mole species. A difference in the length of the centromeric elements between U. talpoides and D. pilirostris might be observed due to certain modifications of the repeating unit.     

Sequence organization and cytological localization of the minor satellite of mouse.

A complete 120 bp genomic consensus sequence for the mouse minor satellite has been determined from enriched L929 centromeric sequences. The extensive sequence homology existing between the major and minor satellite suggests an evolutionary relationship. Some sequences flanking the minor satellite has also been identified and they provide insight into centromeric DNA organization. Isotopic in situ hybridization analysis of the minor satellite to mouse L929 and Mus musculus metaphase spreads showed that this repetitive DNA class is localized specifically to centromeres of all chromosomes of the karyotype. With the use of high resolution non-isotopic fluorescence in situ hybridization the minor satellite is further localized to the outer surface of the centromere in a discrete region at or immediately adjacent to the kinetochore. Our cytological data suggests that the minor satellite might play a role in the organization of the kinetochore region rather than, as previously suggested, sites for general anchoring of the genome to the nuclear matrix.     

Molecular cloning and sequence analysis of cDNA for human transferrin

A cDNA clone for human transferrin was identified from a human liver cDNA library by pre-screening with different ss-cDNA probes against length-fractionated liver mRNAs, positive hybridization-selection and nucleotide sequence analysis. The insert was of 1 kb, encoding human transferrin from aminoacid 403 through the COOH terminus, with a 3' non coding region of 166 nucleotides. This insert hybridized with a single major mRNA species of about 2.4 kb and several genomic DNA restriction fragments. Hybridization of the Southern blots with different parts of the transferrin insert and at different stringences suggest that the various bands observed correspond to splice sites inside one gene rather than to hybridization to several related genes. Finally, a single or a low number of transferrin gene copies seem to exist in the human genome.     

Differential organization of a LINE-1 family in Indian pygmy field mice

Southern blot hybridization analysis of genomic DNAs digested with restriction endonuclease EcoR I and Ava II from Mus musculus domesticus, Mus booduga and Mus terricolor with a cloned repetitive DNA fragment of Mus booduga as a probe showed difference in restriction pattern of this DNA in these three species. Further Southern analysis of the BamH I digested genomic DNAs from these species hybridized with cloned DNA fragment as a probe and sequencing of the cloned DNA revealed that this 252 bp cloned DNA fragment is a part of BamHI repeat element of genus Mus and is 87% homologous to the contiguous portion of the Mus musculus domesticus LINE-1 element. The species specific fragment pattern generated by different restriction endonucleases using this DNA as a probe revealed difference in the organization of LINE-1 repetitive element in the three species of genus Mus.     

Y Chromosome Evolution in the Subgenus Mus (Genus Mus)

A 305 base pair DNA sequence isolated from the Y chromosome of the inbred mouse strain C57BL/10 was used to investigate the pattern and tempo of evolution of Y chromosome DNA sequences for five species in the subgenus Mus, including Mus spretus, Mus hortulanus, Mus abbotti, Mus musculus and Mus domesticus. Variation in hybridization patterns between species was characterized by differences in fragment lengths of both intensely and faintly hybridizing fragments, whereas variation in hybridization patterns within species was characterized primarily by differences in fragment lengths of faintly hybridizing fragments. Phylogenetic analyses were conducted based on fragment size variation within and among species. Phylogenetic relationships inferred from these analyses partly agree with the phylogenetic relationships obtained from biochemical and mitochondrial DNA data. We conclude that a set of DNA sequences common to the Y chromosomes of a closely related group of species in the subgenus Mus has evolved rapidly as reflected by sequence divergence and sequence amplification.     

A Repeated Segment on the Mouse Y Chromosome Is Composed of Retroviral-Related, Y-Enriched and Y-Specific Sequences

We report the isolation and characterization of two recombinant clones containing DNA derived from the Y chromosome of the C57BL/10 inbred mouse strain. Both clones were isolated from a lambda phage library derived from a partial EcoRI digest of C57BL/10 male DNA using the murine retrovirus M720. Characterization of these clones showed they were derived from a repeated segment present on the C57BL/10J Y chromosome that contains sequences found elsewhere in the genome. In addition, one clone contained a sequence, designated YB10, that is unique to the Y chromosome and present in approximately 500 copies on the C57BL/10J Y chromosome. Analysis of Southern blots containing DNAs prepared from females and males of representative species from four subgenera of Mus probed with pYB10 and the 3'LTR from one of the Y-associated retroviruses (MuRVY) revealed that, with the exception of a single fragment observed in both female and male DNA of Mus saxicola, hybridization to pYB10 was observed only to male DNA of the species Mus spretus, Mus hortulanus, Mus musculus, Mus domesticus and Mus abbotti. In addition, the pattern and intensity of hybridization to YB10 and the MuRVY-LTR indicated that sequence of divergence was followed by amplification of Y chromosome sequences containing YB10 and MuRVY. The divergence and amplification occurred separately in each of the ancestral lineages leading to M. spretus, M. hortulanus, M. abbotti, M. musculus and M. domesticus. We suggest that acquisition and amplification of DNA sequences by the mammalian Y chromosome has contributed to its evolution and may imply that the mammalian Y chromosome is evolving at a faster rate than the rest of the genome.     

Isolation, chromosomal localization, and nucleotide sequence of the human HOX 1.4 homeobox

We have isolated a 14-kb DNA sequence containing a single homeobox from a low-stringency screen of a human genomic phage library by using heterologous homeobox sequences as probes. Chromosomal mapping of this clone using in situ hybridization to metaphase chromosomes and a panel of mouse x human somatic cell hybrids localized it to human chromosome 7p13-p15 in the region of the HOX 1 locus. We have sequenced the homeobox and show it has 100% identity to the deduced amino acid sequence of the mouse Hox-1.4 homeobox. We detect no restriction fragment length polymorphisms with the 14-kb clone, which is devoid of any moderately repetitive DNA sequences. This implies an inability of this region to tolerate change in sequence, consistent with a function highly conserved throughout evolution. The regions in the human genome where homeobox-containing loci reside share patterns of organization and sequence and have other gene loci in common, implying evolutionary constraints over these regions and providing clues on how they may have evolved.     

Isolation and characterization of two repetitive DNA fragments located near the centromere of the mouse X chromosome

Two repetitive DNA fragments located on the mouse X chromosome are described. The fragments were isolated from a lambda phage library enriched in X-chromosomal sequences by flow sorting. Both fragments, which are repeated 20 to 50 times in the genome, were mapped to the mouse X chromosome by Southern blot hybridization to DNA from hybrid cells retaining the mouse X chromosome, by dosage analysis, and by in situ hybridization to mouse chromosomes. In mouse strain C57BL/10BK, one fragment appeared to be located only on the X chromosome, while the other fragment had homologous sequences on chromosome 11 in addition to the X chromosome. The latter fragment showed DNA variants between mouse strains, which are potentially useful for mapping. Both fragments cross-hybridized to another mouse species: Mus caroli. In this species, each fragment appeared to be located on the X chromosome, indicating that some X-chromosome repetitive sequences are partially conserved. In addition, one fragment cross-hybridized to human DNA.