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PubMed1990

Development of cloned DNA probes for the identification of snail intermediate hosts within the genus Bulinus.

DNA fragments have been isolated from Bulinus cernicus and cloned in Escherichia coli strain JM109 using pUC18 as the vector. Four recombinant plasmids designated pBC123, pBC126, pBC127 and pBC134 were selected for further analysis and were shown by both dot and Southern blot hybridisations to have different specificities to species representing the four species groups of Bulinus. Non-specific hybridisation occurred with pBC126 whereas pBC123 and pBC134 hybridised only to B. cernicus. Partial restriction maps of the inserts revealed sequence differences between pBC123 and pBC134. Intermediate specificity was shown by pBC127 as this probe hybridised only to DNA from snails of the B. forskalii group and B. truncatus of the B. truncatus/tropicus complex. The results demonstrate the potential of DNA probes for the identification and characterisation of intermediate hosts of schistosomes.

Animals↗
PubMed1990

Characterization of the breakpoint of a t(14;14)(q11.2;q32) from the leukemic cells of a patient with T-cell acute lymphoblastic leukemia.

The leukemic cells and derivative cell line from a 74-year-old male with T-cell acute lymphoblastic leukemia showed chromosomal abnormalities including a t(14;14)(q11.2;q32). This translocation is characteristic of a variety of T-cell malignancies, particularly T-cell prolymphocytic leukemia and the clonal proliferations of peripheral T cells in patients with ataxia-telangiectasia. Using DNA probes that spanned the T-cell receptor alpha chain (TCRA) joining (J) locus, the DNA rearrangement caused by the translocation was identified, cloned, and sequenced. The breakpoint shows site-specific juxtaposition of a TCRA joining segment and DNA from a region of 14q32 centromeric to the immunoglobulin heavy chain locus. Comparison of restriction map and nucleotide sequence from this translocation with other related chromosomal breakpoints suggests a dispersion of breakpoints throughout the 14q32 region.

Amino Acid Sequence↗
PubMed1988

Genetic relatedness of the basic replicon of the virulence plasmid in shigellae and enteroinvasive Escherichia coli.

The regions required for replication (Rep) and for stable maintenance (Stb) of the 230 kilobase virulence plasmid, pMYSH6000, in Shigella flexneri 2a YSH6000 were identified on the restriction enzyme Sa/l fragments, C and O, respectively. The stable plasmid, pMYSH6610, consisting of adjacent fragments C and O was found to belong to the incompatibility group Fl. In all strains of shigellae and enteroinvasive Escherichia coli (EIEC) tested, virulence plasmids were found to be highly homologous with the Rep region of pMYSH6000. The restriction map of this probe was very similar to that of the inc region of RepFIIA replicon. The virulence plasmid of S. flexneri 6, S. boydii, S. dysenteriae and EIEC all coexisted stably with pMYSH6610, whereas that of S. flexneri other than serotype 6 and S. sonnei did not. These findings indicate that all the virulence plasmids of shigellae and EIEC are highly related but can be divided into at least 2 groups.

DNA, Bacterial↗
PubMed1991

Construction and characterization of a NotI linking library of human chromosome 21.

Effective procedures have been developed for constructing NotI linking libraries starting from chromosome-specific genomic libraries. Fifteen different single copy and two rDNA NotI linking clones from human chromosome 21 were identified in two libraries. Their chromosomal origin was confirmed, and regional location established using hybrid cell panels. Hybridization experiments with these probes revealed pairs of genomic NotI fragments, each ranging in size from less than 0.05 to 4.0 Mb. Many fragments displayed cell type variation. The total size of the NotI fragments detected in a human fibroblast cell line (GM6167) and mouse hybrid cell containing chromosome 21 as its only human component (WAV17) were approximately 32 and 34 Mb, respectively. If these fragments were all non-overlapping, this would correspond to about 70% of the 50-Mb content estimated for the whole chromosome. The linking clones will be enormously useful in the subsequent construction of a NotI restriction map of this chromosome. Characterization of these clones indicates the presence of numerous additional sites for other enzymes that recognize sequences containing CpG. Thus most NotI linking clones appear to derive from CpG islands and probably identify the 5' end of genes.

Cell Line↗
PubMed1991

A YAC contig across the fragile X site defines the region of fragility.

The fragile X syndrome is a common cause of mental retardation and is associated with a fragile site at Xq27.3 (FRAXA). Recently, evidence has been presented for the role of methylation and genomic imprinting in the expression of the disease. We have identified a site of methylation in patients by long range restriction mapping of the region. In this paper we present a YAC contig of this area, localise the CpG sequences which are methylated, and show by in situ hybridisation that the site of fragility lies within this region.

Base Sequence↗
PubMed1991

Identification of a common viral integration region in Cas-Br-E murine leukemia virus-induced non-T-, non-B-cell lymphomas.

The Cas-Br-E murine leukemia virus is a nondefective retrovirus that induces non-T-, non-B-cell lymphomas in susceptible NIH/Swiss mice. By using a DNA probe derived from Cas-Br-E provirus-flanking sequences, we identified a DNA region, originally called Sic-1, rearranged in 16 of 24 tumors analyzed (67%). All proviruses were integrated in a DNA segment smaller than 100 bp and were in the same 5'-to-3' orientation. Ecotropic as well as mink cell focus-forming virus types were found integrated in that specific DNA region. On the basis of Southern blot analysis of somatic cell hybrids and progeny of an interspecies backcross, the Sic-1 region was localized on mouse chromosome 9 near the previously described proto-oncogenes or common viral integration sites: Ets-1, Cbl-2, Tpl-1, and Fli-1. Restriction map analysis shows that this region is identical to the Fli-1 locus identified in Friend murine leukemia virus-induced erythroleukemia cell lines and thus may contain sequences also responsible for the development of mouse non-T-, non-B-cell lymphomas.

Animals↗
PubMed1995

Structure of the mouse 3-methyladenine DNA glycosylase gene and exact localization upstream of the alpha-globin gene cluster on chromosome 11.

In this paper we describe the genomic organization of the mouse 3-Methyladenine DNA Glycosylase (MPG) gene and localize three putative regulatory elements around this gene. The MPG gene plays a key role in the excision repair of methylated adenine residues and has been localized upstream of the alpha-globin gene cluster in human and mouse. The human MPG gene has been fully characterized, whereas up to now only the cDNA sequence of the mouse MPG gene had been published. Here, we describe a detailed restriction map, the intron/exon structure, the CpG-rich putative promoter sequence, and the exact localization of the mouse MPG gene with respect to the murine alpha-globin gene cluster. Our analysis reveals a remarkable different exon/intron structure of the mouse MPG gene compared with its human homolog. Two prominent DNase hypersensitive sites (HSS) were found 0.1 and 1.5 kb upstream of the coding sequence. In addition to these elements, an erythroid prominent HSS was mapped at the intron/exon boundary of the last exon. The characterization and localization of the MPG gene in mouse makes it now possible to carry out transgenic and gene targeting experiments and are essential to understand the control of gene expression of the MPG gene in particular and of the whole region in general.

Animals↗
PubMed1991

The ubiquitin-encoding multigene family of flax, Linum usitatissimum.

Ubiquitin (Ubq), a 76-amino acid (aa) protein, is found in all eukaryotic organisms and is one of the most conserved proteins so far studied. It is implicated in many cellular processes. The Ubq-encoding genes (ubq) are generally present as a multigene family. In flax, we have estimated that this multigene family contains at the most ten members. The initial flax ubq sequences were isolated from a flax genomic library in lambda EMBL4 using a heterologous Arabidopsis thaliana ubq probe. An 916-bp fragment from one of the phage clones was subcloned and sequenced. The aa sequence derived from the nucleotide sequence of this fragment is identical to that of other plant Ubqs. This fragment was then used to isolate additional flax ubq clones. In all, eleven phage lambda clones, which represent six members of the gene family, were restriction-mapped and characterized. These six members are represented as three monomers, three poly-Ubqs, one hexamer and two tetramers. They can be present at either a single locus (two of the monomers and one of the poly-Ubqs) or at two loci (the remaining three genes). The other four members of the family are yet to be cloned and characterized.

Amino Acid Sequence↗
PubMed1990

Increased variation in ADH enzyme activity in Drosophila mutation-accumulation experiment is not due to transposable elements at the Adh structural gene.

We present here a molecular analysis of the region surrounding the structural gene encoding alcohol dehydrogenase (Adh) in 47 lines of Drosophila melanogaster that have each accumulated mutations for 300 generations. While these lines show a significant increase in variation of alcohol dehydrogenase enzyme activity compared to control lines, we found no restriction map variation in a 13-kb region including the complete Adh structural gene and roughly 5 kb of both 5' and 3' sequences. Thus, the rapid accumulation of ADH activity variation after 28,200 allele generations does not appear to have been due to the mobilization of transposable elements into or out of the Adh structural gene region.

Alcohol Dehydrogenase↗
PubMed1991

Analysis of Epstein-Barr virus DNA in nude mouse-passaged nasopharyngeal carcinoma tissues.

DNAs extracted from two series of nude mouse-passaged nasopharyngeal carcinoma (NPC) tissues, NPC-295 and -306, and P3HR1 cells were digested with restriction enzyme Bam HI, then electrophoresed on agarose gel and analysed for the Epstein-Barr virus (EBV) DNA fragments by Southern hybridization with 27 radioactive Bam HI DNA fragments and the Eco RI Dhet DNA fragment from cloned EBV DNA of B95-8 cell line. Constant hybridization patterns were revealed in these experiments and the results suggest a stable existence of EBV DNA including replicative form and episomal form in these tissues even after a long time of passage. Neither deletion in YH region nor in I region could be detected when using Bam HI Y, H and I as probes, suggesting the EBV in these tissues are "more complete" viruses. When Bam HI F was used as a probe, an extra Bam HI site was revealed in the analysis of the DNA from NPC-295 suggesting the polymorphism of DNA present in this region. Restriction maps of EBV DNAs in both nude mouse-passaged NPC tissues are proposed. The results suggest that nude mouse-passaged NPC tissue is a good model to study the structure of EBV DNA in tumor tissue.

Adult↗
PubMed1989

A search for c-myb gene regulatory sequences: cloning and restriction analysis of the 18-kb BamHI fragment of chicken chromosomal DNA, containing the 5' part of the c-myb gene.

The 18-kb BamHI fragment of the chicken chromosomal DNA derived from the 5' end of the myb proto-oncogene has been cloned. The 5' part of this clone represented by a 7.2-kb BamHI-EcoRI fragment has been analysed by means of restriction mapping, which revealed the existence of about 2.5 kb of the CpG dinucleotide-rich sequence within this fragment. A short probe prepared from the CpG-rich sequence hybridizes with the 3.6-kb c-myb mRNA. Based on our results and published c-myb cDNA sequence, we conclude that the cloned fragment contains c-myb promoter.

Animals↗
PubMedDate not supplied

[Cloning of genomic sequences of human prointerleukin 1 beta].

The human brain library carried in the EMBL3 vector was employed for isolating prointerleukin 1 beta genomic sequences using three synthetic 20-member oligonucleotides. Oligonucleotides were homologous to the following mRNA regions: 3'-nontranslated region/C1/, 3'-translated region of mRNA/C2/ and the sequence coding N-terminal of mature protein/N1/. The oligonucleotide labeling utilized the terminal nucleotidyltransferase and [alpha-32P] dATP and specific activity of labeled oligonucleotides reached 1.6.10(10) cpm. The sizes of the synthesized labeled sequences (tails) were about 10 b.p. Hybridization probe C1 was used for the first screening and 24 hybridization positive clones were detected. For the next screening probe C2 was used and only 2 hybridization positive clones with different level of hybridization were detected from 24 clones. Probe N1 was used for the third screening and allowed to identify the only positive clone. The characterization by restriction mapping and Southern blot analyses have shown that recombinant phage DNA contains all three exons, coding the mature interleukin 1 beta. Some fragments were recloned to tg130 vector phage and nucleotide sequences of exon 5 (completely) and exon 7, intron 4 and 5 (partially) were determined.

Bacteriophages↗
PubMedDate not supplied

Genetic rearrangements of plasmids containing mtDNA inserts: possible poison sequence.

Restriction mapping of recombinant plasmids indicated the presence of poison sequence(s) in monkey mtDNA. These plasmids were constructed from a 5.2 K.b. BglII mtDNA fragment and pRSVneo or pdel9 as cloning vectors. The poison sequence(s) caused genetic rearrangement of the vectors' nucleotide sequences. Deletion of the suspected poison sequence(s) from the mtDNA fragment increased the transformation efficiency of the produced recombinant plasmids and conserved the vectors' original nucleotide sequences.

Animals↗
PubMed1993

The Streptomyces lividans 66 chromosome contains a 1 MB deletogenic region flanked by two amplifiable regions.

Genetic instability in Streptomyces species often involves large deletions sometimes accompanied by DNA amplification. Two such systems in Streptomyces lividans 66 involve the production of mutants sensitive to chloramphenicol and the production of mutants resistant to the galactose analogue 2-deoxygalactose, respectively. Overlapping cosmids were isolated that span the ca. 1 Mb region between the two amplifiable regions. The structure of the region was confirmed by restriction mapping using the rarely cutting enzymes AseI, BfrI and DraI and pulsed-field gel electrophoresis. The region contains a non-clonable gap flanked by inverted repeats; the structure is consistent with the presence of a physical gap, i.e. a linear chromosome.

Chloramphenicol↗
PubMed1992

Physical linkage of the A-raf-1, properdin, synapsin I, and TIMP genes on the human and mouse X chromosomes.

Genes encoding the neuron-specific phosphoprotein synapsin I (SYN1), the glycoprotein tissue inhibitor of metalloproteinases (TIMP), the proto-oncogene A-raf-1 (ARAF1), and properdin (PFC), a positive regulator of the alternative pathway of human complement, lie within a conserved synteny encompassing the proximal short arm of the human X chromosome (Xp21.1-p11) and the centromeric end of the mouse X chromosome (A1-A5). We have used a mouse interspecific cross to demonstrate genetic linkage of Syn-1, Timp, and Araf and also show physical linkage, with Timp lying only 10 kb from Araf, within an intron of the Syn-1 gene. Detailed restriction mapping shows that Timp is transcribed in the same direction as Araf but in the opposite direction to the Syn-1 gene. Analysis of the corresponding region of the human X chromosome indicates a similar arrangement and in addition shows that the properdin gene lies within 5 kb of the 5' end of the synapsin I gene.

Animals↗
PubMed1998

A new Na+/H+ antiporter, NhaD, of Vibrio parahaemolyticus.

A gene encoding an Na+/H+ antiporter was cloned from chromosomal DNA of Vibrio parahaemolyticus, a slightly halophilic bacterium, and expressed in Escherichia coli cells. The gene enabled mutant E. coli cells, which were unable to grow in the presence of 10 mM LiCl (or 0.2 M NaCl) because of the lack of major Na+(Li+)/H+ antiporters, to grow under such conditions. We detected Na+/H+ antiport activity due to the gene in membrane vesicles prepared from E. coli cells that harbored the plasmid carrying the gene. Li+ was also a substrate for this antiporter. Activity of this antiporter was pH-dependent with highest activity at pH 8.5 to 9 and no activity at 7.0 to 7.5. Restriction mapping and a Southern blot analysis revealed that the cloned gene was different from the nhaA and the nhaB of V. parahaemolyticus. We designated the gene nhaD. The gene was sequenced, and the amino acid sequence of the NhaD protein was deduced. The NhaD is a unique Na+/H+ antiporter with respect to the primary structure compared with known Na+/H+ antiporters.

Amino Acid Sequence↗
PubMed1992

Characterization of two circular plasmids from the marine diatom Cylindrotheca fusiformis: plasmids hybridize to chloroplast and nuclear DNA.

This paper reports the discovery and initial characterization of two small plasmids, pCf1 and pCf2, in the marine diatom Cylindrotheca fusiformis. Extracted diatom DNA separates into two bands in CsCl-Hoechst 33258 dye gradients. Upon agarose gel electrophoresis of a sample of the upper band of the gradient we observed, in addition to high molecular weight (genomic) chloroplast and mitochondrial DNA, pairs of lower molecular weight bands. These bands contained two species of circular plasmid DNA molecules, as shown by electron microscopy. The nucleotide composition of the plasmids, and chloroplast and mitochondrial DNAs is similar, as indicated by their co-banding in the gradients. They were cloned, and their restriction maps determined, showing that pCf1 is 4.27 and pCf2 4.08 kb in size. By hybridization analysis, we showed that pCf1 and pCf2 share regions of similarity, but not identity. Neither plasmid hybridizes with mitochondrial DNA. Both plasmids hybridize with chloroplast DNA, and pCf2 also hybridizes with nuclear DNA.

Cell Nucleus↗
PubMed1991

Structures and organization of major plasma protein genes of the silkworm Bombyx mori.

In the silkworm, Bombyx mori, a group of structurally related proteins, termed 30K proteins, accumulate in the hemolymph of the last instar larvae. We have isolated and characterized three genes, each of which encodes a distinct 30K protein component. Each 30K protein gene is composed of a short first exon and a protein-coding second exon interspersed by a single intron. The transcription initiation site of the 30K protein mRNA was identified at the nucleotide level. A typical TATA box exists some 30 base-pairs upstream from the transcription initiation site. The 5'-flanking region of each gene also contains octamer-like sequences. Restriction mapping analyses revealed that the cloned 46 x 10(3) base-pair region of the chromosomal DNA bears three 30K protein genes. Several copies of highly reiterated retrotransposon-like sequences are present around the 30K protein genes. S1 nuclease protection analysis provided evidence that the biosynthesis of 30K protein is regulated in a stage-specific manner at the transcriptional level in the fat body.

Amino Acid Sequence↗