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PubMed1995

Islands of complex DNA are widespread in Drosophila centric heterochromatin.

Heterochromatin is a ubiquitous yet poorly understood component of multicellular eukaryotic genomes. Major gaps exist in our knowledge of the nature and overall organization of DNA sequences present in heterochromatin. We have investigated the molecular structure of the 1 Mb of centric heterochromatin in the Drosophila minichromosome Dp1187. A genetic screen of irradiated minichromosomes yielded rearranged derivatives of Dp1187 whose structures were determined by pulsed-field Southern analysis and PCR. Three Dp1187 deletion derivatives and an inversion had one breakpoint in the euchromatin and one in the heterochromatin, providing direct molecular access to previously inaccessible parts of the heterochromatin. End-probed pulsed-field restriction mapping revealed the presence of at least three "islands" of complex DNA, Tahiti, Moorea, and Bora Bora, constituting approximately one half of the Dp1187 heterochromatin. Pulsed-field Southern analysis demonstrated that Drosophila heterochromatin in general is composed of alternating blocks of complex DNA and simple satellite DNA. Cloning and sequencing of a small part of one island, Tahiti, demonstrated the presence of a retroposon. The implications of these findings to heterochromatin structure and function are discussed.

Animals↗
PubMed1997

Assessment of genetic diversity among strains of Pseudomonas syringae by PCR-restriction fragment length polymorphism analysis of rRNA operons with special emphasis on P. syringae pv. tomato.

Phylogenetic relationships among 77 bacterial strains belonging to Pseudomonas syringae and Pseudomonas viridiflava species were assessed by analysis of the PCR-restriction fragment length polymorphism (RFLP) patterns of three DNA fragments corresponding to rrs and rrl genes and the internal transcribed spacer, ITS1. No difference among all strains in rrs and rrl genes was observed with 14 restriction enzymes, which confirms the close relationships existing between these two species. The nucleotidic sequence of the internal transcripted spacer (ITS1) between rrs and rrl for the P. syringae pv. syringae strain CFBP1392 was determined. Restriction maps of the PCR-amplified ITS1 region were prepared and compared for all 77 strains. Seventeen RFLP patterns, forming three main clusters, were distinguished. One contained all strains of P. syringae pv. tomato and of other pathovars which had been previously described as closely related by either pathogenicity studies or biochemical analyses. This cluster was equally far from P. viridiflava and from other P. syringae pathovars. These other pathovars of P. syringae formed a less coherent taxon.

DNA, Ribosomal↗
PubMed1995

Long-range organization of reiterated sequences, including the SSX1 cDNA at the OATL1 cluster in Xp11.23.

Analysis of four YACs at the OATL1 locus was undertaken to determine the organization of locally repeated sequences within the OATL1 cluster. A restriction map of ICRFy900C0874, a 600-kb YAC, was constructed using a range of rare-cutting enzymes. Several markers were isolated from the OATL1 cluster, and the YAC map was used for their localization. Markers are shown to be reiterated within the OATL1 cluster, and fine-scale mapping has identified the accurate map position and organization of these repeated sequences within a 275-kb interval. The SSX1 gene involved in synovial sarcoma tumorigenesis and localized at the OATL1 cluster is also shown to be present in a minimum of five copies, all of which map to the aforementioned region. Several markers generated in this study have homologous counterparts at OATL2; they have been utilized to delimit the duplicated region at the OATL clusters. Although instability of one of the OATL1 YACs, ICRFy900F0501, has precluded a precise sizing of this interval, it has been possible to place an upper limit of 520 kb on this region of duplication.

Base Sequence↗
PubMed1991

Characterization of genes encoding hydroxypyruvate reductase in cucumber.

Several clones corresponding to the gene encoding NADH-dependent hydroxypyruvate reductase have been isolated from a cucumber genomic library. Restriction mapping indicates the presence of two HPR genes, hpr-A and hpr-B, in the cucumber genome. Examination of the DNAs of individual plants suggests that hpr-A and hpr-B are most likely alleles at a single locus. The sequence of a 6.7 kb genomic fragment that includes the entire transcribed region, 2.2 kb of 5' flanking sequence, and about 0.8 kb of 3' flanking sequence reveals the presence of 12 introns in hpr-A. These introns are AT-rich relative to the exons. The donor sequence at the 5' end of the sixth intron contains an unusual dinucleotide, GC, rather than the nearly invariant GT. Primer extension analysis maps the transcription initiation site to 61 nucleotides upstream of the translation initiation codon. An AT-rich stretch is centered at position -31 with respect to the transcription initiation site, and a potential CCAAT box is centered at position -138. Several elements that are homologous to regulatory elements of other plant genes have been identified in the flanking regions of hpr-A.

Alcohol Oxidoreductases↗
PubMed1998

Further characterization of the murine collagenase (type IVB) gene promoter and analysis of mRNA expression in murine tissues.

The collagenase B type IV (Col4B) gene is highly expressed in the osteoclast, the primary bone-resorbing cell. However, factors that regulate expression of the Col4B gene are not well characterized. A murine P1 genomic clone containing a 94 kb sequence insert which contains the Col4B gene was isolated. A 4 kb EcoR1 DNA fragment containing the 5' flanking sequence of the gene was further subcloned and restriction mapped. Putative transcription factors such as SRY, Lyf-1, and GATA1 and 2, binding motifs were identified by sequence analysis in this promoter region. Enhancer and suppressor regions were mapped by transient expression of Col4B gene promoter deletion mutant-luciferase reporter gene constructs in HepG2 cells. Col4B mRNA expression in different murine tissues was analyzed by reverse transcription-polymerase chain reaction and demonstrated high levels of expression in bone, clavaria, spleen and thymus. This promoter provides a valuable tool for targeting gene expression to the osteoclast.

Animals↗
PubMed1989

Cloning and characterization of a cluster of linked Bacillus subtilis late competence mutations.

We characterized a segment of chromosomal DNA from Bacillus subtilis that was required for the development of genetic competence. The chromosomal DNA was cloned from a group of genetically linked and phenotypically similar Tn917lac insertion mutants deficient in competence. This cluster of mutations defined the comG locus. Chromosomal DNA flanking each of the six insertions was cloned. Restriction maps of the cloned plasmids revealed that their chromosomal inserts consisted of overlapping fragments. These data, together with Southern blots of chromosomal DNA from the comG mutants, showed that the six Tn917lac comG insertions occurred in the following order: comG12, comG39, comG412, comG107, comG56, and comG210. Expression of the comG Tn917lac insertions was from a promoter located upstream from the first insertion, comG12. This was determined genetically and by low-resolution S1 nuclease mapping of the 3' terminus. The comG region spanned about 5 kilobase pairs, based on low-resolution S1 nuclease mapping of the transcription terminator and Northern blotting. The comG12 mutation had a partial epistatic effect on the expression of one other com locus, comE, but none of the other comG mutations affected expression of this or any other com gene tested. Based on these conclusions, and on its size and phenotype, the comG locus must be organized as a polycistronic operon that is subject to competence-specific regulation.

Bacillus subtilis↗
PubMed1992

Molecular cloning and partial characterization of a parrot papillomavirus.

The genome of a papillomavirus isolated from a cutaneous lesion on the head of an African grey parrot (Psittacus erithacus timneh) was cloned into pBR322, and a restriction map was prepared. Several short portions of the DNA were sequenced allowing the genome to be aligned with HPV-la. In Southern blot hybridizations, under conditions of low and medium stringency (Tm-40 and -33 degrees), but not at a higher stringency, this viral DNA annealed weakly with only 1 of 17 mammalian papillomavirus genomes tested. Furthermore, PePV DNA hybridized with the DNA from the European chaffinch only at low stringency, indicating that it represents a unique avian papillomavirus.

Animals↗
PubMed1991

Low basal transcription of genes for tissue-specific collagens by fibroblasts and lymphoblastoid cells. Application to the characterization of a glycine 997 to serine substitution in alpha 1(II) collagen chains of a patient with spondyloepiphyseal dysplasia.

Cultured dermal fibroblasts were shown, using amplification of cDNA by the polymerase chain reaction, to produce very low levels of spliced transcripts from the COL2A1 gene that encodes the cartilage-specific alpha 1(II) chains of type II collagen. Cultured lymphoblastoid cells were also shown to produce very low levels of spliced transcripts from the COL1A1 and COL1A2 genes that encode the alpha 1(I) and alpha 2(I) chains of type I collagen, the COL2A1 gene that encodes type II collagen, and the COL3A1 gene that encodes the alpha 1(III) chains of type III collagen. Amplified cDNAs prepared from lymphoblastoid cells were used to identify previously characterized heterozygous mutations in the COL1A1 and COL1A2 genes from two patients with osteogenesis imperfecta and in the COL3A1 gene from a patient with the Ehlers-Danlos syndrome type IV. Amplified alpha 1(II) cDNA from fibroblasts and lymphoblastoid cells of a child with spondyloepiphyseal dysplasia congenita was also used to localize sequence mismatches using chemical modification of cDNA:cDNA heteroduplexes by hydroxylamine and cleavage with piperidine. The amplification products containing the mismatched region were sequenced and the mutation was shown to change the codon GGC for glycine 997 to AGC for serine in the triple helical domain of the alpha 1(II) chains. The corresponding region of the genomic DNA was sequenced and the heterozygous point mutation was shown to be in exon 48 of the COL2A1 gene. Allelic restriction mapping showed that neither parent carried the mutation in their leucocytes. This mutation emphasizes the importance of COL2A1 mutations in producing the spondyloepiphyseal dysplasia phenotype. The low basal rate of transcription ("illegitimate transcription"), splicing, and polyadenylation of tissue-specific mRNAs by cultured dermal fibroblasts and lymphoblastoid cells provides the opportunity to localize and sequence mutations in amplified cDNA in patients from whom affected tissue is unavailable.

Base Sequence↗
PubMed1992

Physical and biochemical characterization of the cloned LYS5 gene required for alpha-aminoadipate reductase activity in the lysine biosynthetic pathway of Saccharomyces cerevisiae.

The LYS5 and LYS2 genes of Saccharomyces cerevisiae are required for the synthesis of alpha-aminoadipate reductase in the lysine pathway. The LYS5 gene, originally cloned as a DNA insert of the plasmid pSC5, has been subcloned on a 3.2 kb SphI-Sau3AI DNA fragment of the recombinant plasmid pSR7. An internal 2.1 kb HpaI-HpaI DNA fragment of the subclone, upon Southern hybridization, exhibits homology with HpaI-restricted wild-type S. cerevisiae genomic DNA. The lys5+ transformants exhibited alpha-aminoadipate reductase activity similar to that of wild-type cells. S1 nuclease analysis localizes the transcription initiation site relative to the detailed restriction map, and reveals the direction of transcription, as well as the transcript size of the LYS5 gene which can be no greater than 1.65 kb. From this it is estimated that the encoded polypeptide is appreciably smaller than the 4 kb LYS2 gene product. These results provide a physical and biochemical characterization of the cloned LYS5 gene. Based on these observations, it is concluded that the LYS5 gene encodes a relatively small polypeptide of the large heteropolymeric alpha-aminoadipate reductase.

Aldehyde Oxidoreductases↗
PubMed1994

Detection and species identification of Cryptosporidium oocysts using a system based on PCR and endonuclease restriction.

The polymerase chain reaction (PCR) was used to produce a 556 bp nucleotide stretch, employing primers based on the published sequence of the 18S rRNA genes in Cryptosporidium parvum and C. muris. This sequence was found to contain 3 Mae I endonuclease restriction sites, 1 of which was present only in C. parvum. Mae I restriction of PCR products from 2 C. parvum isolates (one of human origin and the other of bovine origin), 1 C. muris isolate, and 1 C. baileyi isolate, showed a specific and reproducible profile for C. parvum that was different from the one obtained for both C. muris and C. baileyi. From these data, new Mae I restriction maps were proposed for the three species. The system was then used to screen 6 C. parvum isolates (from human and bovine hosts), and the C. parvum-specific profile was obtained for all isolates examined. It should be possible to adapt this protocol to detect small numbers of C. parvum oocysts in environmental samples (e.g. in water supplies).

Animals↗
PubMed1993

Paramutation, an allelic interaction, is associated with a stable and heritable reduction of transcription of the maize b regulatory gene.

The b gene of maize encodes a transcriptional activator of anthocyanin pigment biosynthetic genes. Certain b alleles undergo paramutation: a unidirectional, heritable alteration of one allele caused by the presence of another allele. B-I (intensely pigmented plant) is always changed to B' (weakly pigmented plant) in the B'/B-I heterozygote, such that all progeny receive the B' allele. The "new" B', which was B-I in the previous generation, is weakly pigmented and fully capable of changing another B-I allele into B'. It was not previously known whether paramutation is associated with altered b expression, altered B protein function or both. Our results show that B' acts in trans to suppress the transcription of B-I, with transcription remaining low in subsequent generations, even when the original B' allele segregates away. The products of B-I and B' are equally capable of activating the transcription of their target genes, indicating they are functionally equivalent. Genomic restriction maps, DNA sequence and methylation of B' and B-I were compared. Despite dramatic differences in phenotype and transcription of B' and B-I, no evidence for rearrangements, changes in sequence or changes in methylation was found. These results provide no support for models involving "dominant negative" proteins, gene conversion or transposable element interactions. We suggest that b paramutation involves a physical interaction between the alleles that suppresses transcription and promotes a change in chromatin structure that is heritable.

Alleles↗
PubMed1991

Novel mercury resistance determinants carried by IncJ plasmids pMERPH and R391.

HgCl2 resistance (Hgr) in a strain of Pseudomonas putrefaciens isolated from the River Mersey was identified as plasmid-borne by its transfer to Escherichia coli in conjugative matings. This plasmid, pMERPH, could not be isolated and was incompatible with the chromosomally integrated IncJ Hgr plasmid R391. pMERPH and R391 both express inducible, narrow-spectrum mercury resistance and detoxify HgCl2 by volatilization. The cloned mer determinants from pMERPH (pSP100) and R391 (pSP200) have very similar restriction maps and express identical polypeptide products. However, these features show distinct differences from those of the Tn501 family of mer determinants. pSP100 and pSP200 failed to hybridize at moderate stringency to merRTPA and merC probes from Tn501 and Tn21, respectively. We conclude that the IncJ mer determinants are only distantly related to that from Tn501 and its closely homologous relatives and that it identifies a novel sequence which is relatively rare in bacteria isolated from natural environments.

Cloning, Molecular↗
PubMed1991

Characterization of the sugarcane streak agent as a distinct geminivirus.

The relationship between maize streak virus and the geminivirus causing streak in sugarcane was investigated. The DNA of sugarcane streak virus does not cross-hybridize detectably with that of maize streak virus and vice versa. Restriction mapping of native replicative form viral DNA (genome size 2.7 kb) and of cloned viral DNA, combined with limited sequencing and estimated DNA sequence divergence, showed that sugarcane streak virus is as unrelated to maize streak virus and digitaria streak virus as these are different from each other. The virus is only distantly related to wheat dwarf virus and chloris striate mosaic virus. Based on these results, we propose that the agent causing sugarcane streak is a distinct geminivirus.

Blotting, Western↗
PubMed1992

Transformation of the mycoparasite Parasitella simplex to neomycin resistance.

The facultatively parasitic zygomycete Parasitella simplex was transformed to neomycin resistance by a vector, which had been developed primarily for transformation of its host Absidia glauca. This plasmid, pAmNEF21, contained the bacterial resistance gene for neomycin (NPTII) under the control of the promoter region from the gene for elongation factor 1 (tef) isolated from A. glauca. Both flanking regions of the marker gene contain parts of the structural tef gene. DNA isolated from two Parasitella transformants was re-transformed in E. coli and the resulting plasmids, pAt21 and pAt35, were analyzed. The restriction map and Southern blot analysis show that both plasmids are rearranged. They had lost the structural tef information and were found to contain new DNA fragments, which were identical in both cases. Southern blot analysis of the transformants indicates that the rearranged plasmids are present in the fungal transformants and that the changes are not the result of re-transformation in E. coli. Plasmids were only recovered after growth under selective conditions. Southern blot analysis and re-transformation with undigested transformant DNA shows that the plasmids are replicated autonomously.

Blotting, Southern↗
PubMed1994

Mutagenesis of the paracrystalline surface protein array of Aeromonas salmonicida by endogenous insertion elements.

The tetragonal paracrystalline surface protein array (A-layer) of the fish pathogenic bacterium Aeromonas salmonicida is a virulence factor and bacteria which are unable to produce A-layer are attenuated in their ability to kill fish. Ten independent mutants of Aeromonas salmonicida which were unable to produce A-layer were isolated by growth at 30 degrees C. These mutants displayed either reduced synthesis of the A-layer subunit, synthesis of a truncated subunit, or complete loss of the ability to produce the subunit protein. Restriction mapping and analysis by polymerase chain reaction showed that the mutations had resulted from insertion of two different insertion sequence (IS) elements into different sites in the A-layer subunit gene (vapA) and its promoter. Sequence comparisons indicated that ISAS1 is unique among reported IS elements. It is 1223 bp long with imperfect terminal inverted repeats of 22 bp and insertion resulted in a duplicated 8 bp target sequence in vapA. ISAS1 expressed a 42,000 molecular weight (M(r)) protein in mini-cells. ISAS2 was 1084 bp long, expressed proteins of M(r) 38,000 and 39,000 in vitro, had imperfect 29 bp terminal inverted repeats and had duplicated a 3 bp target sequence. Sequence comparisons indicated that ISAS2 was also unique to A. salmonicida; however, the proteins encoded by ISAS2 showed strong homology to the putative transposases encoded by the IS30 family of IS elements. Southern analyses showed that both ISAS1 and ISAS2 were restricted to A. salmonicida strains A449 and A450 where they were present in low copy number. The ability of these two IS elements to mutate the ability of A. salmonicida to produce its paracrystalline surface array provides a novel method for the attenuation of virulence.

Aeromonas↗
PubMed1994

Structure and evolution of the largest chloroplast gene (ORF2280): internal plasticity and multiple gene loss during angiosperm evolution.

We have determined the nucleotide sequence of the Pelargonium x hortorum ORF2280 homolog, the largest gene in the plastid genome of most land plants, and compared it to published homologs from Nicotiana tabacum, Epifagus virginiana, Spinacia oleracea, and Marchantia polymorpha. Multiple alignment of protein sequences requires an extraordinary number of gaps, indicating a very high frequency of insertion/deletion events during the evolution of the protein; however, the overall predicted size of the protein varies relatively little among the five species. At 2,109 codons, the Pelargonium gene is smaller than other land plant ORF2280 homologs and exhibits a rate of nucleotide substitution several times higher relative to Nicotiana, Epifagus, and Spinacia. Southern-blot and restriction-mapping studies were carried out to uncover length variation in ORF2280 homologs from 279 species (representing 111 families) of angiosperms. In many independent angiosperm lineages, this gene has sustained deletions ranging in size from 200 bp to almost 6 kb. Based on the severity of deletions, we postulate that the chloroplast homolog of ORF2280 has become nonfunctional in at least four independent lineages of angiosperms.

Amino Acid Sequence↗
PubMed1991

Cloning and expression in Escherichia coli of dextranase genes from Bacteroides thetaiotaomicron.

A genomic bank was constructed in Escherichia coli HB101, consisting of DNA fragments from Bacteroides thetaiotaomicron strain 489 inserted within the vector pBR322. By screening on complex medium containing blue dextran, 10 stable dextranase-positive (Dex+) clones were isolated. Seven groups of Dex+ inserts were identified on the basis of their restriction maps and hybridization responses. Dextanase activity of the recombinant clones was weak, and was revealed on the selection medium after 15 days. Subcloning of a Sau3AI partially digested 3.2-kb insert in the expression vector pDR720 greatly enhanced dextranse activity on blue dextran plates in one clone, but the delay remained unaltered. This suggested that the enzyme was released by cell lysis. Expression of this 0.7-kb subcloned insert was dependent on the promoter region of tryptophan operon carried by pDR720.

Bacteroides↗
PubMed1993

A comparison of the multiple alleles of xylS carried by TOL plasmids pWW53 and pDK1 and its implications for their evolutionary relationship.

Both of the independently isolated TOL plasmids pWW53 and pDK1 contain multiple regions homologous to the xylS regulatory gene of the archetypal TOL plasmid pWW0. The three homologues on pWW53 vary in the extent of their homology to xylSpWW0, xylS1pWW53 is 99% identical to xylSpWW0 and is located relative to the single copy of xylRpWW53 in exactly the same way as xylS and xylR on pWW0. The DNA sequence of xylS3pWW53 is 87% identical to the xylSpWW0 sequence within the coding region but the non-coding DNA upstream is not homologous. There is a frame-shift change at the end of the coding region which causes the C terminus of XylS3pWW53 to be extended by an additional 10 amino acids relative to XylSpWW0. xylS2pWW53 is anomalous and appears to encode a truncated pseudogene lacking the first 525 bases found in the other xylS genes. Evidence is presented to show that both xylS1pWW53 and xylS3pWW53 act as regulators of meta pathway operons. Plasmid pDK1 carries two homologues of xylS. xylS1pDK1 is functional and is a hybrid gene: its 5' end and the upstream sequences are highly homologous to both xylS1pWW53 and xylSpWW0, whereas its 3' end is identical to xylS3pWW53. The sequence of xylS2pDK1 is identical to that of the anomalous truncated xylS2pWW53. Comparison of the organization and the restriction maps of the xyl catabolic operons on pDK1 and pWW53, together with the nucleotide sequences presented here, indicates that the catabolic DNA on pDK1 has derived from a replicon on which the xyl genes are organized similarly to pWW53 and that a genetic rearrangement has taken place involving a reciprocal recombination internal to two of its xylS homologues.

Alleles↗