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PubMed1993

Genome analysis of adenovirus 4 isolated over a six year period.

Genome analysis was carried out on 74 adenovirus 4 (Ad4) isolates from patients in Manchester between 1984 and 1989. Most of the isolates were associated with conjunctivitis. Of the 74 isolates studied, 51 were Ad4a and 10 were Ad4p (the prototype strain). The remaining isolates consisted of two new genome types we have designated Ad4a2 (10 isolates) and Ad4a3 (3 isolates). Most of the genome types co-circulated during the period of study. The Bst E II and Xho I restriction maps of the new variants are presented and compared with those of Ad4p. We are unable to associate genome types with particular clinical presentations.

Adenovirus Infections, Human↗
PubMed1989

The isolation and comparison of cellulase genes from two strains of Ruminococcus albus.

Endo-1,4-beta-glucanase genes have been cloned from two strains of Ruminococcus albus recently isolated in this laboratory. Although the strains were phenotypically similar, cross-hybridization studies between them showed significant genetic differences, with only 20% of the genome forming DNA heteroduplexes. Heteroduplexes displayed an average dissociation temperature 9 degrees C lower than that of the homoduplex. Consistent with this, restriction maps of the two endoglucanase genes showed no similarity, and hybridization work using the endoglucanase genes as probes revealed that neither gene was present in the genome of the other isolate of R. albus. Comparative enzyme characterization showed differences between the enzymes in their response to temperature, pH and substrate preference.

Animals↗
PubMed1994

Distribution of membrane bound guanylyl cyclases in human intestine.

The quantification and distinction of particulate guanylyl cyclases in the human intestine were considered by an enzymatic approach, which comprised the signal transduction from receptor binding to cGMP formation, and, in addition, by showing the expression of an intracellular portion of these transmembrane proteins. Basal guanylyl cyclase (GC) activities were 50 to 80 pmol cGMP formation/min/mg protein and were stimulated up to twofold by heat stable enterotoxin, but were not significantly influenced by atrial natriuretic factor. Enzymatic analysis of colonoscopic specimens pointed to the prevalence of guanylyl cyclase C in the terminal ileum and in the large bowel including colon ascendens, colon descendens, sigmoid, and rectum. The availability of sequence information on human guanylyl cyclases permitted the development of a polymerase chain reaction approach for distinguishing the expression of GC-A and GC-C in human tissue samples. The expression levels of particulate guanylyl cyclases found by polymerase chain reaction in surgical biopsy specimens confirmed the enzymatic data, in that substantial expression of GC-C was found not only in the small intestine but also in the large bowel. According to the restriction mapping of amplificates, GC-C prevailed over GC-A throughout the human intestine, particularly in the mucosal layers.

Adult↗
PubMed1994

The human type 2 cystatin gene family consists of eight to nine members, with at least seven genes clustered at a single locus on human chromosome 20.

The family of type 2 cystatin proteins are a class of cysteine proteinase inhibitors found in a variety of human fluids and secretions, where they appear to provide protective functions. To establish the size of the human gene family encoding these proteins, we isolated cosmid and lambda genomic clones. Restriction mapping, partial sequence analysis, and hybridization studies identified a total of seven distinct genes, six of which correspond to known genes and proteins. Sequence analysis showed that the seventh gene, CSTP2, is an apparent pseudogene carrying a nonsense mutation in exon 1 distinct from that in CSTP1. Southern blots of genomic DNA probed with gene-specific probes accounted for all but one or two sets of fragments containing exon 1, and one or two sets of fragments containing exon 3, indicating that the human type 2 cystatin gene family consists of eight or nine members. Southern blot analysis of large restriction fragments using these gene-specific probes indicates that all seven of the cloned type 2 cystatin genes are clustered at a single locus on human chromosome 20. This locus is no larger than about 910 kb, and possibly as small as 365 kb. We designate this as the CST locus and suggest a numbering system for the cystatin genes.

Amino Acid Sequence↗
PubMed1993

Origins of polymorphism at a polypurine hypervariable locus.

We present characterisation of a hypervariable locus, D8S210, mapped to the telomeric region of the short arm of chromosome 8. The locus is highly polymorphic with alleles varying in size from 1.8 kb to 24 kb. Sequence data from 7 alleles shows that the variable region is entirely polypurine on one strand with a tetranucleotide repeating unit GGAA at the margins and diverged versions of this motif internally. The margins are conserved between alleles; polymorphism occurring in the internal regions of the repeat. Alleles are inherited in a Mendelian manner and one new mutation has been observed in analysis of 51 meioses. Use of single copy flanking sequences to elaborate the polymorphism revealed loss of single copy DNA in 3 unrelated families and in 2 other unrelated individuals. Restriction mapping shows that this loss is similar for different sized alleles in all three families suggesting that it was an early event that may have involved a flanking Alu sequence. We present evidence that the polypurine region can adopt triplex conformations in vitro. Such structures may facilitate loss or gain of unique sequences in the genome, contribute to mutation at conformation transition points and drive the hypervariability (> 99% heterozygosity) of this locus.

Alleles↗
PubMed1993

DNA sequence and transcript analysis of transposon MuA2, a regulator of Mutator transposable element activity in maize.

The 4942 bp DNA sequence of Zea mays transposon MuA2 was determined. Previous evidence indicated MuA2 controls activity of the Mu1 transposon located in the mutable allele a1-mum2. MuA2 contains two large, ATG-initiated open reading frames (ORFs) of 612 and 232 codons, respectively, located on opposite strands. MuA2 produces two transcripts, each containing one of these ORFs. Four different tandem direct repeat sequences are located downstream of the 612 codon ORF. The restriction map of MuA2 is identical to that of transposon MuR1, which also is known to regulate mutability of a1-mum2. Furthermore, except for a single nucleotide, MuA2 is identical to the Mutator element Mu9.

Amino Acid Sequence↗
PubMed1989

Episomal HPV 16 DNA isolated from a cervical carcinoma presents a partial duplication of the early region.

An invasive cervical carcinoma was found to harbor an episomal variant of human papillomavirus (HPV) type 16 DNA, with a size of about 10.1 kb. A genomic library of the tumor was constructed in bacteriophage lambda and a recombinant phage clone was isolated by screening with HPV 16 probe. Analysis by restriction mapping and Southern hybridization showed that the isolate contained a 2.2 kb duplication of the early region, which included part of E6, all E7 and part of E1 open reading frames. Possible consequences of this duplication for oncogenesis are discussed.

DNA Probes, HPV↗
PubMed1999

Characterisation of a 5.5-kb cryptic plasmid present in different isolates of Bacteroides spp. originating from Hungary.

The plasmid profiles of 97 Bacteroides isolates collected during screening for different pathogenic markers of this genus were investigated. In all, 48% of 69 isolates from infections that belonged to six species harboured low mol.wt plasmids (2.8-11.0 kb). Similar plasmids were also found in 39% of 28 isolates, belonging to eight species, from faeces of healthy persons. The two most frequently obtained types were the 5.5- and the 4.2-kb plasmids, which were present in 70% and 52% of all plasmid-bearing isolates, respectively. Restriction endonuclease analysis revealed that the 5.5-kb plasmids found in the different Bacteroides spp. exhibited the same restriction map, with the exception that pBVP61 lacked the PstI recognition site. The two plasmid types (4.2 and 5.5 kb) seem to be most widely distributed among Bacteroides isolates independent of the site of isolation and with some differences depending on geographic regions.

Bacterial Adhesion↗
PubMed1993

Rapid analysis of -alpha 3.7 thalassaemia and alpha alpha alpha anti 3.7 triplication by enzymatic amplification analysis.

In this report we describe a PCR-based method for the diagnosis of the most common form of alpha thalassaemia, the -alpha 3.7 deletion which occurs throughout all tropical and subtropical regions of the world. The same procedure also identifies the reciprocal recombinant chromosome (alpha alpha alpha anti 3.7). Restriction mapping of the PCR products has enabled us to distinguish between the type I (-alpha 3.7 I), type II (-alpha 3.7 II) and type III (-alpha 3.7 III) deletions. This strategy will be very useful in screening programmes of alpha thalassaemia occurring on its own or in association with beta thalassaemia and sickle cell disease.

Globins↗
PubMed1991

Genetic analysis of the rfb region of Shigella flexneri encoding the Y serotype O-antigen specificity.

The gene cluster (rfb region) which determines the biosynthesis of the Shigella flexneri O-antigen of the Y serotype specificity was cloned from a S. flexneri serotype 2a strain. Two plasmids, pPM2212 and pPM2213, which conferred O-antigen biosynthesis were generated from separate cosmid clones by deletion with Clal. These plasmids expressed O-antigen in Escherichia coli K12 like that of the parental strain, as assessed by reactions to antisera in colony and Western immunoblots, sensitivity to bacteriophage Sf6, and by silver staining of lipopolysaccharides separated by sodium dodecyl sulphate/polyacrylamide gel electrophoresis. These plasmids also mediated O-antigen expression in an E. coli K12 rfb-delete background, indicating that all the necessary genes have been cloned. A detailed restriction map of the region has been constructed and analysis of various subclones has allowed the limits of the coding region for O-antigen biosynthesis to be defined to a maximum of 11 kb. Expression of these plasmids demonstrates a novel phenotype associated with control of lipopolysaccharide chain length. The gene(s) responsible maps adjacent to, but separate from, those associated with the biosynthesis of the O-antigen unit. Analysis of plasmid-encoded proteins in minicells and maxicells has facilitated the construction of a physical map. Finally, plasmid pPM-2212 was used to probe a collection of S. flexneri serotypes by Southern hybridization. With the exception of serotype 6, which appears to be unrelated, a similar pattern was found in all serotypes.

Blotting, Southern↗
PubMed1990

cDNA clones encoding immunoglobulin lambda chains from rabbit expressing the phenotype c7.

A cDNA library derived from spleen cells of an unimmunized rabbit expressing the c7 phenotype of Ig lambda chains (c7+, c21-) was screened with V lambda or C lambda probes of a lambda light chain bearing c21 epitopes. The nucleotide sequences of three hybridizing clones were found to be identical within the V lambda, J lambda and C lambda regions. The V lambda region was 97% similar to that of the functional germ-line gene V lambda 2, and the C lambda region was identical to that of gene C lambda 6, recently identified. Gene C lambda 6 exhibited four codon differences when compared with gene C lambda 5, the latter encoding c21 epitopes. The data presented here and in the accompanying report (Jaton, J.-C. et al., Eur. J. Immunol. 1990, 20:2713) support the view that gene C lambda 6 encodes the C region of c7 lambda chains and that c7 and c21 markers designate two distinct isotypic forms of lambda chains. On the basis of comparative Southern blotting analyses and restriction maps of cloned genomic regions containing V lambda and C lambda genes, a scheme is proposed to account for the c7- and c21- phenotypes.

Amino Acid Sequence↗
PubMed1991

Chromosome rearrangement in Leishmania mexicana M379.

Circular extrachromosomal elements were observed in a variety of Leishmania species. We show here that two lines originating from the same isolate have been found to contain a circular DNA molecule of 26.6 kb and a linear chromosome of about 250 kb, respectively, which share a homology of more than 20 kb. The circular DNA molecule and its related region on the linear chromosome were cloned and their restriction maps compared. This investigation reveals information about chromosome rearrangement in L. mexicana M379. Further examination will enable us to understand the nature of chromosome rearrangement such as circularization or linearization.

Animals↗
PubMed1996

Integration of the DNA of a novel filamentous bacteriophage VSK from Vibrio cholerae 0139 into the host chromosomal DNA.

An unusual filamentous bacteriophage, VSK, containing single-stranded, circular DNA as its genome was isolated from Vibrio cholerae 0139 strains P07 and B04. Unlike other single-stranded DNA phages, VSK can integrate its genome into the chromosome of the host and enter into a lysogenic state. The double-stranded replicative form (RF) of the single-stranded phage DNA was isolated. A restriction map of the VSK RF DNA was constructed using HaeII, AvaII, ClaI and XbaI. By Southern blot analysis of the chromosomal DNA of the lysogen using labeled phage DNA as probe, the attachment site (attP) on the viral genome was also identified.

Bacteriophages↗
PubMed1989

rhs gene family of Escherichia coli K-12.

Two additional members of a novel Escherichia coli gene family, the rhs genes, have been cloned and characterized. The structures of these loci, rhsC and rhsD, have been compared with those of rhsA and rhsB. All four loci contain a homologous 3.7-kilobase-pair core. Sequence comparison of the first 300 nucleotides of the cores showed that rhsA, rhsB, and rhsC are closely related, with only 1 to 2% sequence divergence, whereas rhsD is 18% divergent from the others. The beginning of the core coincides with the initiation of an open reading frame that extends beyond the 300 nucleotides compared. Whether a protein product is produced from this open reading frame has not been established. However, nucleotide substitutions which differentiate the cores have highly conservative effects on the predicted protein products; this suggests that products are made from the open reading frame and are under severe selection. The four rhs loci have been placed on both the genetic and restriction maps of E. coli K-12. A fifth rhs locus remains to be characterized. In terms of size, number, and sequence conservation, the rhs genes make up one of the most significant repetitions in E. coli, comparable to the rRNA operons.

Amino Acid Sequence↗
PubMed1991

Direct selection: a method for the isolation of cDNAs encoded by large genomic regions.

We have developed a strategy for the rapid enrichment and identification of cDNAs encoded by large genomic regions. The basis of this "direct selection" scheme is the hybridization of an entire library of cDNAs to an immobilized genomic clone. Nonspecific hybrids are eliminated and selected cDNAs are eluted. These molecules are then amplified and are either cloned or subjected to further selection/amplification cycles. This scheme was tested using a 550-kilobase yeast artificial chromosome clone that contains the EPO gene. Using this clone and a fetal kidney cDNA library, we have achieved a 1000-fold enrichment of EPO cDNAs in one cycle of enrichment. More significantly, we have further investigated one of the "anonymous" cDNAs that was selectively enriched. We confirmed that this cDNA was encoded by the yeast artificial chromosome. Its frequency in the starting library was 1 in 1 x 10(5) cDNAs and after selection comprised 2% of the selected library. DNA sequence analysis of this cDNA and of the yeast artificial chromosome clone revealed that this gene encodes the beta 2 subunit of the human guanine nucleotide-binding regulatory proteins. Restriction mapping and hybridization data position this gene (GNB2) to within 30-70 kilobases of the EPO gene. The selective isolation and mapping of GNB2 confirms the feasibility of this direct selection strategy and suggests that it will be useful for the rapid isolation of cDNAs, including disease-related genes, across extensive portions of the human genome.

Base Sequence↗
PubMed1990

Physical mapping of new DNA probes near the fragile X mutation (FRAXA) by using a panel of cell lines.

The fragile X syndrome is a very common disorder, but there has been little progress toward isolating the fragile X mutation (FRAXA). We describe a panel of 14 somatic cell hybrid lines, lymphoblastoid cell lines, and peripheral lymphocytes with X-chromosome translocation or deletion breakpoints near FRAXA. The locations of the breakpoints were defined with 16 established probes between pX45d (DXS100) and St14-1 (DXS52). Seven of the cell lines had breakpoints between the probes RN1 (DXS369) and U6.2 (DXS304), which flank FRAXA at distances of 3-5 centimorgans. The panel of cell lines was used to localize 16 new DNA probes in this region. Six of the probes-VK16, VK18, VK23, VK24, VK37, and VK47--detected loci near FRAXA, and it was possible to order both the X-chromosome breakpoints and the probes in relation to FRAXA. The order of probes and loci near FRAXA is cen-RN1,VK24-VK47-VK23-VK16,FRAXA-++ +VK21A-VK18-IDS-VK37-U6.2-qter. The breakpoints near FRAXA are sufficiently close together that probes localized with this panel can be linked on a large-scale restriction map by pulsed-field gel electrophoresis. This panel of cell lines will be valuable in rapidly localizing other probes near FRAXA.

Animals↗
PubMed1994

Complete structure of the gene encoding an immunodominant antigen of Dirofilaria immitis and larva-specific synthesis of primary transcript.

The complete gene encoding an immunodominant antigen of Dirofilaria immitis was isolated from a Charomid 9-36 genomic DNA library. This genomic DNA clone termed 'Dg2' was characterized by restriction mapping, DNA sequencing of the 5' flanking region, the exon/intron boundaries and the polyadenylation addition site. The Dg2 with 4872 bp in length consisted of five exons interspersed with four introns. These exons reveal a single open reading frame followed by a long 3' non-coding region of 1383 bp. The open reading frame of 969 bp encodes a polypeptide of 322 amino acids with a molecular weight of 34,400. The ATG translation initiation codon starts 22 nucleotides downstream from the 5' end of the first exon. The polyadenylation signal sequence. AATAAA, is located at the 3' end of the last exon. The transcription initiation site was determined by primer extension technique. S1 nuclease mapping analysis demonstrated that the primary transcript derived from Dg2 is synthesized in microfilariae but not in male or female adult worms. The result suggests that the stage-specific expression of Dg2 is regulated at the level of primary transcript.

Amino Acid Sequence↗
PubMed1992

Activation of EVI1 gene expression in human acute myelogenous leukemias by translocations spanning 300-400 kilobases on chromosome band 3q26.

Retroviral activation of Evi-1 gene expression is one of the most common transforming events in murine myeloid leukemias. To evaluate the role of the EVI1 gene in human acute myelogenous leukemia (AML), leukemic blasts or cell lines from 116 patients were examined. In eight patients the EVI1 gene was expressed and all but one had cytogenetically detectable translocations of chromosome 3q26 where the EVI1 gene has been localized. To identify breakpoints, a restriction map that spans 1700 kilobases (kb) of the EVI1 locus was developed by pulsed-field gel electrophoresis. In one case, t(3;3)(q21;q26), a rearrangement was localized to 170-330 kb 5' of the gene. In a second case, t(3;3)(q21;q26), there was a rearrangement 13 kb 5' of the gene. This rearrangement was cloned and shown to be due to the fusion of sequences from 3q21-22 with the EVI1 locus. In the third case, ins(3)-(q21q25q27), there was a rearrangement that mapped 150 kb downstream from the 5' end of the gene.

Base Sequence↗