Characterization of squid crystallin genes. Comparison with mammalian glutathione S-transferase genes.

Previous experiments have indicated that the crystallins of the squid lens (S-crystallins) are evolutionarily related to glutathione S-transferases (GST) (EC 2.5.1.18). Here we confirm by peptide sequencing that the crystallins of the lens of the squid Ommastrephes sloani pacificus comprise a family of GST-like proteins. Squid lens extracts showed 400 times less GST activity than those of liver using 1-chloro-2,4-dinitrobenzene as a substrate, suggesting that the abundant GST-like crystallins lack enzymatic activity. Four different cDNAs (pSL20-1, pSL18, pSL11, and pSL4) showed 20-25% similarity in homologous regions with mammalian GST polypeptides. pSL20-1, pSL18, and pSL4 each encode an S-crystallin with a unique internal peptide that is unrelated to mammalian GSTs or any other sequence in GenBank. The S-crystallin family is encoded in a minimum of 9-10 genes, and the exon-intron structures of at least two of these (SL20-1 and SL11) are similar to those of the mammalian GST genes. The SL20-1 gene has six exons, with the its unique internal peptide encoded precisely in exon 4; the SL11 gene lacks a unique internal peptide and has five exons. Experiments using bacterial chloramphenicol acetyltransferase as a reporter gene showed that at least 84 and 111 base pairs of 5'-flanking sequence are needed for function of the SL20-1 and SL11 promoters, respectively, in a transfected rabbit lens epithelial cell line (N/N1003A). Within these regions each has a putative TATA box and an upstream AP-1 site overlapping with antioxidant responsive-like elements, which are regulatory elements in the rat GST Ya and quinone reductase genes responsive to oxidative stress.     

Recombination between compatible plasmids containing homologous segments requires the Bacillus subtilis recE gene product.

Plasmid pSL103 was previously constructed by cloning a Trp fragment (approximately 2.3 X 10(6) daltons) from restriction endonuclease EcoRI-digested chromosome DNA of Bacillus pumilus using the neomycin-resistance plasmid pUB110 (approximately 2.8 X 10(6) daltons) as vector and B. subtilis as transformation recipient. In the present study the EcoRI Trp fragment from pSL103 was transferred in vitro to EcoRI fragments of the Bacillus plasmid pPL576 to determine the ability of the plasmid fragments to replicate in B. subtilis. Endonuclease EcoRI digestion of pPL576 (approximately 28 X 10(6) daltons) generated three fragments having molecular weights of about 13 X 13(6) (the A fragment), 9.5 X 10(6) (B fragment, and 6.5 X 10(6) (C fragment). Trp derivatives of pPL576 fragments capable of autonomous replication in B. subtilis contained the B fragment (e.g., pSL107) or both the B and C fragments (e.g., pSL108). Accordingly, the B fragment of pPL576 contains information essential for autonomous replication. pSL107 and pSL108 are compatible with pUB110. Constructed derivatives of the compatible plasmids pPL576 and pUB110, harboring genetically distinguishable EcoRI-generated Trp fragments cloned from the DNA of a B. pumilus strain, exhibited relatively high frequency recombination for a trpC marker when the plasmid pairs were present in a recombination-proficient strain of B. subtilis. No recombination was detected when the host carried the chromosome mutation recE4. Therefore, the recE4 mutation suppresses recombination between compatible plasmids that contain homologous segments.     

Identification of the minimal replication region of the multicopy Streptomyces plasmid pSL1

The 1.52-kb minimal replication origin of the 3.9-kb Streptomyces plasmid pSL1 was determined using a bifunctional derivative, pMCP44, of pSL1. Plasmids with linker insertions into the pSL1 part of pMCP44 were isolated from Escherichia coli. The sites of insertion were determined by restriction enzyme analysis and the ability of the mutant plasmids to replicate in S. lividans 66 was determined. All except one of the inserts in the 1.52-kb essential region inactivated replication. A 104-bp segment from this region could function as a replication origin in the presence of a helper plasmid containing a nonoverlapping pSL1 fragment. The sequence of this 104-bp fragment shows similarities to those of known plasmid replication origins.     

PCR with 5-methyl-dCTP replacing dCTP.

When dCTP is replaced by methyl5-dCTP in the polymerase chain reaction some templates cannot be efficiently amplified by Taq polymerase or Vent polymerase using standard cycling parameters. However, this phenomenon can be overcome by increasing the temperature of the denaturation steps to 100 degrees C, or by adding dITP to destabilize the m5dC:dG base pairs. Once the block to amplification of m5dC-substituted DNA was overcome, methylated DNA from the 'superpolylinker' of the plasmid pSL 1180 was used as a substrate to check the methyl-sensitivity of a variety of restriction endonucleases. The m5dC-substituted DNAs should also be valuable substrates for defining the specificity of methyl-dependent endonucleases.     

Mu insertion duplicates a 5 base pair sequence at the host inserted site.

Nucleotide sequences were analyzed across the two ends of lysogenic Mu DNA. These ends were cloned separately in lambdapMu hybrid particles that derived from a single Mu lysogen in the lac Z part of lambdaplac5. The obtained data imply that Mu lysogenization was associated with the duplication of 5 base pairs present in lac DNA at the Mu insertion site. As a result of this duplication, Mu DNA is flanked by two copies of five identical base pairs oriented as direct repeats. A similar conclusion has been obtained independently by other investigators with the use of a different Mu lysogen (D. Kamp and R. Kahmann, personal communication). Thus Mu insertion seems to have a striking similarity to typical IS-mediated insertions that were found to be associated with a short DNA duplication at the target site.     

Identification, cloning and sequencing of the replication region of Lactococcus lactis ssp. lactis biovar. diacetylactis Bu2 citrate plasmid pSL2

The replication region of the 7.8 kilobase (kb) citrate plasmid pSL2 from Lactococcus lactis ssp. lactis biovar. diacetylactis Bu2 was identified. Deletion derivatives of pSL2 were introduced into plasmid-free strain Bu2-60 and tested for their ability to replicate autonomously. The region necessary for replication was identified by comparison of the pSL2 derivatives, cloned and sequenced. No homologies were detected by comparing the putative Rep protein of pSL2 with replicons of other plasmids of Gram-positive bacteria. A part of an IS-element flanking the replication region was found.     

The cloning and expression of the Staphylococcus aureus enterotoxin A gene in Escherichia coli cells

Gene ent-A has been cloned on phage vector pSL5 with the use of the gene library of S. aureus FR1722(H). It is located within DNA fragment Hind III having 2,500 nucleotide pairs.     

Pheromone-responsive conjugative vancomycin resistance plasmids in Enterococcus faecalis isolates from humans and chicken feces

The drug resistances and plasmid contents of a total of 85 vancomycin-resistant enterococcus (VRE) strains that had been isolated in Korea were examined. Fifty-four of the strains originated from samples of chicken feces, and 31 were isolated from hospital patients in Korea. Enterococcus faecalis KV1 and KV2, which had been isolated from a patient and a sample of chicken feces, respectively, were found to carry the plasmids pSL1 and pSL2, respectively. The plasmids transferred resistances to vancomycin, gentamicin, kanamycin, streptomycin, and erythromycin to E. faecalis strains at a high frequency of about 10(-3) per donor cell during 4 hours of broth mating. E. faecalis strains containing each of the pSL plasmids formed clumps after 2 hours of incubation in broth containing E. faecalis FA2-2 culture filtrate (i.e., the E. faecalis sex pheromone), and the plasmid subsequently transferred to the recipient strain in a 10-min short mating in broth, indicating that the plasmids are responsive to E. faecalis pheromones. The pSL plasmids did not respond to any of synthetic pheromones for the previously characterized plasmids. The pheromone specific for pSL plasmids has been designated cSL1. Southern hybridization analysis showed that specific FspI fragments from each of the pSL plasmids hybridized with the aggregation substance gene (asa1) of the pheromone-responsive plasmid pAD1, indicating that the plasmids had a gene homologous to asa1. The restriction maps of the plasmids were identical, and the size of the plasmids was estimated to be 128.1 kb. The plasmids carried five drug resistance determinants for vanA, ermB, aph(3'), aph(6'), and aac(6')/aph(2'), which encode resistance to vancomycin, erythromycin, kanamycin, streptomycin, and gentamicin/kanamycin, respectively. Nucleotide sequence analyses of the drug resistance determinants and their flanking regions are described in this report. The results described provide evidence for the exchange of genetic information between human and animal (chicken) VRE reservoirs and suggest the potential for horizontal transmission of multiple drug resistance, including vancomycin resistance, between farm animals and humans via a pheromone-responsive conjugative plasmid.     

Features of two hepatitis B virus (HBV) DNA integrations suggest mechanisms of HBV integration.

Two integrated hepatitis B virus (HBV) DNA molecules were cloned from two primary hepatocellular carcinomas each containing only a single integration. One integration (C3) contained a single linear segment of HBV DNA, and the other integration (C4) contained a large inverted duplication of viral DNA at the site of a chromosome translocation (O. Hino, T.B. Shows, and C.E. Rogler, Proc. Natl. Acad. Sci. USA 83:8338-8342, 1986). Sequence analysis of the virus-cell junctions of C3 placed the left virus-cell junction at nucleotide 1824, which is at the 5' end of the directly repeated DR1 sequence and is 6 base pairs from the 3' end of the long (L) negative strand. The right virus-cell junction was at nucleotide 1762 in a region of viral DNA (within the cohesive overlap) which shared 5-base-pair homology with cellular DNA. Sequence analysis of the normal cellular DNA across the integration site showed that 11 base pairs of cellular DNA were deleted at the site of integration. On the basis of this analysis, we suggest a mechanism for integration of the viral DNA molecule which involves strand invasion of the 3' end of the L negative strand of an open circular or linear HBV DNA molecule (at the DR1 sequence) and base pairing of the opposite end of the molecule with cellular DNA, accompanied by the deletion of 11 base pairs of cellular DNA during the double recombination event. Sequencing across the inverted duplication of HBV DNA in clone C4 located one side of the inversion at nucleotide 1820, which is 2 base pairs from the 3' end of the L negative strand. Both this sequence and the left virus-cell junction of C3 are within the 9-nucleotide terminally redundant region of the HBV L negative strand DNA. We suggest that the terminal redundancy is a preferred topoisomerase I nicking region because of both its base sequence and forked structure. Such nicking would lead to integration and rearrangement of HBV molecules within the terminal redundancy, as we have observed in both our clones.     

Site-specific in vitro binding of plasmid pUB110 to Bacillus subtilis membrane fraction.

The in vitro membrane binding of pSL103, a composite plasmid consisting of Staphylococcus aureus plasmid pUB110 and a Bacillus pumilus trpC+ DNA fragment, to the Bacillus subtilis membrane fraction was studied with a total lysate of B. subtilis cells. The binding reaction required a heat treatment at 45 degrees C and had an optimum KCl concentration of 60 mM. Nonradioactive pSL103, but not Escherichia coli plasmid pACYC184, competed with 3H-labeled pSL103 for binding to the membrane. By the use of 32P-labeled restriction fragments of pSL103 and pUB110, it has been found that only the pUB110 portion of pSL103 binds to the membrane and that there are four specific regions in pUB110 which bind to the membrane. Two of the four binding regions flank the replication origin. This in vitro binding was high-salt sensitive and apparently independent of the configurations of the plasmid. We have previously shown that the functional product of the initiation gene dna-1 is required in vivo both for replication initiation and the binding of a DNA region near the replication origin to the membrane. Unlike in vivo binding, which is high-salt resistant and dependent on the product of dna-1 gene (type-I binding), the in vitro binding reported in this paper was high-salt sensitive and independent of the dna-1 gene product (type-II binding).     

Genetic characterization of an Escherichia coli mutant deficient in organophosphonate biodegradation

An E. coli mutant deficient in organophosphonate biodegradation has been complemented with a cosmid library prepared from a BamHI partial digest of wild-type E. coli W3110. Mutant E. coli SL724, when transformed with cosmid pSL163 and plasmid pSL263, regained the ability to exploit ethylphosphonate as a sole source of phosphorus during growth. In route to complementation, the Tn5 insert of SL724 was subcloned and restriction enzyme mapped. Complementing pSL163 and pSL263 were also characterized via restriction enzyme digests.     

Tandem duplication. A novel type of triplet repeat instability

Triplet repeat sequence (TRS) inserts containing (CTG.CAG)(n) (17-175 units in length) were tandemly duplicated when propagated in plasmids in Escherichia coli. The products of this novel type of TRS genetic instability are tracts of as many as 34 multiple units, which contain the entire TRS as well as 129 base pairs of nonrepetitive flanking sequence. The duplication process required the presence of two or more TRS-containing units. Close proximity (170 base pairs) of the TRS to the R6K gamma origin of replication of the pUTminiTn5Cm-derived constructs stimulated the tandem duplication process. These events are proposed to occur due to secondary structure formation, stalling of DNA synthesis, and slippage-mediated misalignment of the complementary strands relative to each other during DNA replication. This mechanism may account for the TRS-associated duplications in protein kinase and metalloprotease genes in neuroblastomas and melanomas, as well as the massive repeat expansions in type II triplet repeat neurological diseases.     

Diversity in a chorion multigene family created by tandem duplications and a putative gene-conversion event

Summary Two families of high-cysteine chorion proteins inBombyx mori are encoded in 15 tandemly arranged nonidentical gene pairs. It is assumed that this locus arose by duplication with subsequent sequence divergence. We have compared DNA sequences from two such neighboring pairs of genes in an attempt to understand the manner in which diversity has been generated and/or removed. A high level of sequence identity (91%–99%) was found between the repeats throughout the transcribed and flanking regions, with two significant exceptions. First, in the DNA segment encoding a conserved region of the chorion proteins, ten substitutions were detected in a 39-base-pair region. This localized region of high variability would suggest an intergene conversion-like event. Second, a length difference of 141 base pairs was detected in a region encoding the carboxy-terminal arm of the protein. This difference can be explained by three separate reiterations of single codons (3 base pairs) separated in time by duplication or triplication events.     

Discordance in Variation of the ITS Region and the Mitochondrial COI Gene in the Subterranean Amphipod Crangonyx islandicus

The amphipod Crangonyx islandicus is a recently discovered species endemic to Iceland. Populations of C. islandicus are highly structured geographically and genetically. The COI and 16S mitochondrial genes confine six monophyletic groups which have diverged for up to 5 million years within Iceland, and may present two cryptic species. To investigate the potential cryptic species status we analyse here the internal transcribed spacers (ITS1 and ITS2) and compare its variation with the patterns obtained with the mtDNA. The ITS regions present much less divergence among the geographic regions in comparison with the mtDNA, distances based on ITS1 are correlated with the COI distances as well as with geographic distances, but most of the variation is observed within individuals. The variation in the ITS region appears to have been shaped both by homogenization effect of concerted evolution and divergent evolution. A duplication of 269 base pairs is found in the ITS1 of all individuals from the southern populations, its divergence from its paralog appears to predate the split of the different groups within Iceland but some evidence point to rapid diversification after the split. This duplication does not affect the secondary structures found in the 3' and 5' ends of the sequence, suggested to have a role in the excision of the ITS1. Compensatory base changes within the ITS2 sequences which have been suggested to be a species indicator were not detected.     

Molecular characterization of a nonautonomous transposable element (dTph1) of petunia.

An insertion sequence of 283 base pairs has been isolated from the DFR-C gene (dihydroflavonol-4-reductase) of petunia. This insert was found only in a line unstable for the An1 locus (anthocyanin 1, located on chromosome VI) and not in fully pigmented progenitor and revertant lines or in stable white derivative lines. This implies that the An1 locus encodes the DFR-C gene. The unstable An1 system in the line W138 is known to be a two-element system, the autonomous element being located on chromosome I. In the presence of the autonomous element, W138 flowers exhibit a characteristic pattern of red revertant spots and sectors on a white background. In the absence of the autonomous element, the W138 allele gives rise to a stable recessive (white) phenotype. Sequence analysis of progenitor, unstable, and revertant alleles revealed dTph1 to contain perfect terminal inverted repeats of 12 base pairs. In DFR-C, it is flanked by an 8-base pair target site duplication. Sequences homologous to dTph1 are present in at least 50 copies in the line W138. Sequence analysis of An1 revertant alleles indicated that excision, including removal of the target site duplication, is required for reversion to the wild-type phenotype. Derivative stable recessive alleles showed excision of dTph1 and a rearrangement of the target site duplication. dTph1 is the smallest transposable element described to date that is still capable of transposition. The use of dTph1 in tagging experiments and subsequent gene isolation is discussed.     

两种高效 RNA 干涉载体系统的构建及应用

在真核细胞基因功能研究中, RNA 干涉 (RNAi) 已成为一种强有力的选择性沉默基因表达的实验工具. 建立一套可在哺乳动物培养细胞中高效、经济地表达 siRNA 的载体系统是 RNA 干涉研究的必要前提之一. 从 HepG2 细胞基因组 DNA 中克隆得到 H1 全长启动子 (374 bp),以之为基础构建了两套 RNA 干涉载体系统, pSL 和带有绿色荧光蛋白 (EGFP) 标签的 pESL ,并对 p53 基因进行了相应的 RNA 干涉研究. 干涉质粒瞬时转染 HepG2 细胞后,分别利用半定量 RT-PCR 和蛋白质印迹检测 p53 表达水平. 与商品化载体 pSilencer^TM 3.1-H1 hygro 相比, pSL 和 pESL 对 p53 基因表达具有更高的干涉效率. 结果显示：干涉载体 pSL 和 pESL 能高效特异地下调目的基因表达,可作为哺乳动物中基因功能分析的有效工具.     

Nucleotide sequences and properties of the sites involved in lysogenic insertion of the bacteriophage HP1c1 genome into the Haemophilus influenzae chromosome.

Bacteriophage HP1c1 lysogenizes its host Haemophilus influenzae Rd by inserting its genome into the bacterial chromosome. The DNA segments corresponding to the integration regions on the phage and host chromosomes and the two junctions formed between phage and host sequences on lysogenic insertion were isolated and propagated in Escherichia coli HB101 as hybrid plasmids by using pBR322 as the vector. The nucleotide sequences in the vicinity of the point of recombinational insertion were determined. Phage and host DNA shared an extensive, nearly identical, segment that was 183 base pairs long. This segment consisted of 93 identical residues and a 27-residue portion containing 6 mismatches, followed by 63 identical residues. Recombinational insertion occurred within the 63-residue identical segment and involved neither duplication nor deletion of any residues. Short inverted repeats consisting of clustered A-T base pairs were present within the two 27-residue segments. Two additional sites on the host chromosome showed significant hybridization to the phage-host homology region.     

Transcriptional divergence of the duplicated oxidative stress-responsive genes in the Arabidopsis genome

Previous studies have indicated that Arabidopsis thaliana experienced a genome-wide duplication event shortly before its divergence from Brassica followed by extensive chromosomal rearrangements and deletions. While a large number of the duplicated genes have significantly diverged or lost their sister genes, we found 4222 pairs that are still highly conserved, and as a result had similar functional assignments during the annotation of the genome sequence. Using whole-genome DNA microarrays, we identified 906 duplicated gene pairs in which at least one member exhibited a significant response to oxidative stress. Among these, only 117 pairs were up- or down-regulated in both pairs and many of these exhibited dissimilar patterns of expression. Examination of the expression patterns of PAL1 and PAL2, ACD1 and ACD2, genes coding for two Hsp20s, various P450s, and electron transfer flavoproteins suggests Arabidopsis evolved a number of distinct oxidative stress response mechanisms using similar gene sets following the duplication of its genome.     

Recent duplication of the common carp (Cyprinus carpio L.) genome as revealed by analyses of microsatellite loci

Genome duplications may have played a role in the early stages of vertebrate evolution, near the time of divergence of the lamprey lineage. Additional genome duplication, specifically in ray-finned fish, may have occurred before the divergence of the teleosts. The common carp (Cyprinus carpio) has been considered tetraploid because of its chromosome number (2n = 100) and its high DNA content. We studied variation using 59 microsatellite primer pairs to better understand the ploidy level of the common carp. Based on the number of PCR amplicons per individual, about 60% of these primer pairs are estimated to amplify duplicates. Segregation patterns in families suggested a partially duplicated genome structure and disomic inheritance. This could suggest that the common carp is tetraploid and that polyploidy occurred by hybridization (allotetraploidy). From sequences of microsatellite flanking regions, we estimated the difference per base between pairs of alleles and between pairs of paralogs. The distribution of differences between paralogs had two distinct modes suggesting one whole-genome duplication and a more recent wave of segmental duplications. The genome duplication was estimated to have occurred about 12 MYA, with the segmental duplications occurring between 2.3 and 6.8 MYA. At 12 MYA, this would be one of the most recent genome duplications among vertebrates. Phylogenetic analysis of several cyprinid species suggests an evolutionary model for this tetraploidization, with a role for polyploidization in speciation and diversification.     

Pheromone-Responsive Conjugative Vancomycin Resistance Plasmids in Enterococcus faecalis Isolates from Humans and Chicken Feces

The drug resistances and plasmid contents of a total of 85 vancomycin-resistant enterococcus (VRE) strains that had been isolated in Korea were examined. Fifty-four of the strains originated from samples of chicken feces, and 31 were isolated from hospital patients in Korea. Enterococcus faecalis KV1 and KV2, which had been isolated from a patient and a sample of chicken feces, respectively, were found to carry the plasmids pSL1 and pSL2, respectively. The plasmids transferred resistances to vancomycin, gentamicin, kanamycin, streptomycin, and erythromycin to E. faecalis strains at a high frequency of about 10⁻³ per donor cell during 4 hours of broth mating. E. faecalis strains containing each of the pSL plasmids formed clumps after 2 hours of incubation in broth containing E. faecalis FA2-2 culture filtrate (i.e., the E. faecalis sex pheromone), and the plasmid subsequently transferred to the recipient strain in a 10-min short mating in broth, indicating that the plasmids are responsive to E. faecalis pheromones. The pSL plasmids did not respond to any of synthetic pheromones for the previously characterized plasmids. The pheromone specific for pSL plasmids has been designated cSL1. Southern hybridization analysis showed that specific FspI fragments from each of the pSL plasmids hybridized with the aggregation substance gene (asa1) of the pheromone-responsive plasmid pAD1, indicating that the plasmids had a gene homologous to asa1. The restriction maps of the plasmids were identical, and the size of the plasmids was estimated to be 128.1 kb. The plasmids carried five drug resistance determinants for vanA, ermB, aph(3′), aph(6′), and aac(6′)/aph(2′), which encode resistance to vancomycin, erythromycin, kanamycin, streptomycin, and gentamicin/kanamycin, respectively. Nucleotide sequence analyses of the drug resistance determinants and their flanking regions are described in this report. The results described provide evidence for the exchange of genetic information between human and animal (chicken) VRE reservoirs and suggest the potential for horizontal transmission of multiple drug resistance, including vancomycin resistance, between farm animals and humans via a pheromone-responsive conjugative plasmid.