Isolation and characterization of a mouse subtelomeric sequence

A mouse subtelomeric sequence, ST1, was generated from genomic DNA of the mouse HR9 (129/Sv origin) cell line by the polymerase chain reaction (PCR) using a single telomeric primer. ST1 was cloned and characterized: it is composed of 670 bp of novel DNA sequence flanked on each end by inverted telomeric hexanucleotide repeats (TTAGGG)_n. PCR amplification from BALB/c mouse DNA using this single primer gave the same major product. Southern analysis and PCR using internal ST1 primers confirmed that the ST1 sequence is present in mouse genomic DNA. In situ hybridization to metaphase chromosomes of SJL origin mapped ST1 to many, if not every, mouse telomere. PCR experiments using different combinations of the telomeric, minor satellite, and ST1 primers indicated that some ST1 copies are adjacent to minor satellite sequences, that telomeric and ST1 sequences are not generally interspersed with minor satellite sequences,and that ST1 and the minor satellite have a consistent and specific orientation relative to each other and to the telomere.by H.F. Willard     

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.     

Cloning and molecular characterization of a novel chromosome specific centromere sequence of Chinese hamster.

We isolated and characterized the first chromosome-specific satellite DNA (HC2sat) of Chinese hamster. This novel satellite was localized to the pericentric region of hamster chromosome 2. The 2.8 kb long repeat unit of HC2sat was identified and two such units were sequenced. Extended short range periodicity could not be revealed in repeat units. These elements are amongst the largest satellite repeat units reported from mammals to date. HC2sat is a major constituent of the pericentric region of CHO chromosome 2 representing a 7-14 Mb long DNA segment. Studies of long range organization of HC2sat indicated that the formation of the satellite array might occur in different phases and involved different amplification mechanisms.     

Isolation and characterization of polymorphic simple sequence repeat loci in Armillaria ostoyae

A modified hybridization strategy was used to construct a microsatellite enriched library from DNA of Armillaria ostoyae, a serious root pathogen on pine. Sequence characterization of 19 random clones revealed 12 distinct loci harbouring a repetitive motif. Primer design from the flanking regions allowed for their development as polymerase chain reaction based markers. Polymorphic assessment at both the population and global levels revealed levels of variation useful for genetic studies. The level of cross‐species amplification observed with closely related Armillaria species was high, raising the possible exploitation of these primers across the genus.     

Sequence, higher order repeat structure, and long-range organization of alpha satellite DNA specific to human chromosome 8.

We have characterized alphoid repeat clones derived from a chromosome 8 library. These clones are specific for human chromosome 8, as demonstrated by use of a somatic cell hybrid mapping panel and by in situ hybridization. Hybridization of the clones to HindIII digests of human genomic DNA reveals a complex pattern of fragments ranging in size from 1.3 to greater than 20 kb. One clone, which corresponds in size to the most prevalent genomic HindIII fragment, appears to represent a major higher order repeat in the chromosome 8 centromere. The DNA sequence of this clone reveals a dimeric organization of alphoid monomers. Restriction analysis of two other clones indicates that they are derivatives of this same repeat unit. The chromosome 8 alphoid clones hybridize to EcoRI fragments of genomic DNA ranging up to 1000 kb in length and reveal a high degree of polymorphism between chromosomes. Distribution of higher order repeat units across the centromere was examined by two-dimensional gel electrophoresis. Repeat units of the same size class tended to cluster together in restricted regions of centromeric DNA.     

Isolation and initial characterization of a bacterial consortium able to mineralize fluorobenzene.

Fluorinated compounds are known to be more resistant to microbial degradation than other halogenated chemicals. A microbial consortium capable of aerobic biodegradation of fluorobenzene (FB) as the sole source of carbon and energy was isolated by selective enrichment from sediments collected in a drain near an industrial site. A combination of three microbial strains recovered from the enriched consortium was shown to be necessary for complete FB mineralization. Two of the strains (F1 and F3) were classified by 16S rRNA analysis as belonging to the Sphingobacterium/Flavobacterium group, while the third (F4) falls in the beta-Proteobacteria group, clustering with Alcaligenes species. Strain F4 was consistently found in the liquid cultures in a much greater proportion than strains F1 and F3 (86:8:6 for F4, F1, and F3, respectively). Stoichiometric release of fluoride ions was measured in batch and fed-batch cultures. In batch cultures, the consortium was able to use FB up to concentrations of 400 mg liter(-1) and was able to utilize a range of other organic compounds, including 4-fluorophenol and 4-fluorobenzoate. To our knowledge this is the first time biodegradation of FB as a sole carbon source has been reported.     

Isolation and initial characterization of the mouse Dnmt3l gene

We have isolated and sequenced the mouse zinc finger gene, Dnmt3l (DNA cytosine-5-methyltransferase 3-like), on mouse chromosome 10, showing similarity to members of the DNMT3/Dnmt3 family. The Dnmt3l protein contains an ADD zinc finger, which Dnmt3l shares with other Dnmt3 family members and Atrx. RT-PCR analysis showed Dnmt3l expression in testis, thymus, ovary, and heart, as well as in 7-day, 15-day, and 17-day mouse embryos.     

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.     

Isolation and analysis of DNA markers specific to human chromosome 15.

Chromosome-specific DNA markers provide a powerful approach for studying complex problems in human genetics and offer an opportunity to begin understanding the human genome at the molecular level. The approach described here for isolating and characterizing DNA markers specific to human chromosome 15 involved construction of a partial chromosome-15 phage library from a human/Chinese hamster cell hybrid with a single human chromosome 15. Restriction fragments that identified unique- and low-copy loci on chromosome 15 were isolated from the phage inserts. These fragments were regionally mapped to the chromosome by three methods, including Southern analysis with a mapping panel of cell hybrids, in situ hybridization to metaphase chromosomes, and quantitative hybridization or dosage analysis. A total of 42 restriction fragments of unique- and low-copy sequences were identified in 14 phage. The majority of the fragments that have been characterized so far exhibited the hybridization pattern of a unique locus on chromosome 15. Regional mapping assigned these markers to specific locations on chromosome 15, including q24-25, q21-23, q13-14, q11-12, and q11. RFLP analysis revealed that several markers displayed polymorphisms at frequencies useful for genetic linkage analysis. The markers mapped to the proximal long arm of chromosome 15 are particularly valuable for the molecular analysis of Prader-Willi syndrome, which maps to this region. Polymorphic markers in this region may also be useful for definitively establishing linkage with one form of dyslexia. DNA probes in this chromosomal region should facilitate molecular structural analysis for elucidation of the nature of instability in this region, which is frequently associated with chromosomal aberrations.     

Isolation and characterization of simple sequence repeat loci in the gray tree frog, Hyla chrysoscelis.

A gray tree frog (Hyla chrysoscelis) genomic library was constructed and characterized with regard to the incidence and complexity of simple sequence repeat (SSR) loci. The partial genomic library, containing approximately 10,000 clones with an average-sized insert of 350 bp, was screened with six SSR repeat oligonucleotides (AC, AG, ACG, AGC, AAC, and AAG). Screening identified 31 unique positive clones containing 41 SSR loci. Sequences of tandemly arrayed dinucleotide repeats were more common (36 of 41) than trinucleotide repeats. Twenty-six loci were identified using the AC dinucleotide probe, while 7 loci were identified using the AG dinucleotide probe. An additional 3 AT dinucleotide loci were serendipitously identified. The AT repeats generally comprised the longest dinucleotide repeat loci. The SSR repeat loci reported here should provide potent markers for identity, parentage, and short-lineage determinations in large-scale experiments using gray tree frogs.     

Isolation and nucleotide sequence of a mouse histidine tRNA gene.

We have sequenced a 1307 base pair mouse genomic DNA fragment which contains a histidine tRNA gene. The sequence of the putative mouse histidine tRNA differs from the published sequence of sheep liver histidine tRNA by a single base change in the D-loop. It does not contain an unpaired 5' terminal G residue, as reported for Drosophila and sheep histidine tRNAs. The gene does not contain introns. The 3' flanking region contains a typical RNA polymerase III termination site of 6 consecutive T residues. 523 residues after the 3' end of the his tRNA coding region, the mouse DNA contains a sequence 72% homologous to part of the consensus sequence of the B1 (alu) family.     

Isolation and sequence of a cDNA encoding mouse IMP dehydrogenase.

Inosinic acid (IMP) dehydrogenase (IMPD) catalyzes the conversion of IMP to XMP as the first committed step in GMP biosynthesis de novo. We have isolated a cDNA containing the complete coding region of mouse IMPD by its ability to complement a bacterial mutant lacking IMPD activity. Two independent cDNA clones were isolated by complementation, of which the longest was 1.7 kb in length. Northern analyses, using the IMPD cDNA as a probe, indicated that mature IMPD mRNA was a single species approx. 2.0 kb in size. Mouse IMPD is almost identical to Chinese hamster and human IMPDs and is highly conserved between Escherichia coli and mouse, with a direct amino acid (aa) identity of 39%, which increases to 60% if conserved aa are considered. The leader region of our longest cDNA clone is G + C-rich and contains two tandem copies of a G + C-rich direct repeat.     

Isolation and characterization of a mouse protein C cDNA.

Protein C (PC) is a vitamin K-dependent serine protease, a deficiency of which results in thrombus. There is no spontaneously occurring mouse model of the disease. Attempts to create such a model in mice by using anti-sense gene technology requires isolation of a normal mouse PC cDNA. When a mouse liver (BALB/c) cDNA library was screened using a human PC cDNA as a probe, nine overlapping cDNA clones were isolated and sequenced. The cloned mouse PC cDNA comprised 1,512 nucleotides and the open reading frame of the cDNA encoded a polypeptide of 461 amino acids residues including a leader peptide composed of 41 amino acids. Mouse PC exhibited high homology to both human and bovine PCs. Mouse PC also had several structural features common in other PCs; locations of 23 Cys residues, location of putative beta-hydroxy Asp71, possible carbohydrate attachment sites involving Asp residues at amino acid positions 249, 314, and 330, and location of active sites such as His212, Asp258, and Ser361. Northern blot hybridization analysis identified a single species of mouse PC mRNA (2.0 kb in length) in mouse liver.     

Isolation and characterization of a 25-kilodalton protein from mouse testis: sequence homology with a phospholipid-binding protein.

A 25-kDa epididymal secretory protein (MEP 9), isolated from mouse epididymal fluid, has recently been characterized in our laboratory [Rankin et al., Biol Reprod 1992; 46:747-766]. The polyclonal antibody raised against this protein was found to recognize a 25-kDa component in epididymal fluid and testicular extract. The 25-kDa testicular antigen (MTP) was purified by means of ammonium sulfate precipitation, gel filtration, and anion-exchange chromatography; MTP was found to be similar to MEP 9 in several properties including molecular mass (25 kDa), isoelectric point (pI 6.0), and immunoreactivity when the proteins were resolved in the presence of SDS (one-dimensional and two-dimensional PAGE). However, when the proteins were resolved under non-denaturing conditions, MTP showed strong immunoreactivity while MEP 9 did not. This observation suggests that although the 25-kDa antigens from the epididymal fluid and testicular extract are quite similar, they may have different immunological conformations. When analyzed for amino acid composition and partial amino acid sequence, the testicular antigen showed substantial homology (> 80%) with a phosphatidylethanolamine-binding protein characterized from bovine brain. MTP also showed phosphatidylethanolamine-binding activity (Kd = 1.95 x 10(-5) M, Bmax = 1.86 nmol/micrograms MTP), suggesting that the mouse 25-kDa protein is a member of the phospholipid-binding protein family and may have a role in lipid metabolism during sperm maturation.