Aminoglycoside-Resistance Mechanisms in Multidrug-Resistant Staphylococcus aureus Clinical Isolates

Aminoglycoside resistance in six clinically isolated Staphylococcus aureus was evaluated. Genotypical examination revealed that three isolates (HLGR-10, HLGR-12, and MSSA-21) have aminoglycoside-modifying enzyme (AME) coding genes and another three (GRSA-2, GRSA-4, and GRSA-6) lacked these genes in their genome. Whereas isolates HLGR-10 and HLGR-14 possessed bifunctional AME coding gene aac(6′)-aph(2′′), and aph(3′)-III and showed high-level resistance to gentamycin and streptomycin, MSSA-21 possessed aph(3′)-III and exhibited low resistance to gentamycin, streptomycin, and kanamycin. The remaining three isolates (GRSA-2, GRSA-4, and GRSA-6) exhibited low resistance to all the aminoglycosides because they lack aminoglycoside-modifying enzyme coding genes in their genome. The transmission electron microscopy of the three isolates revealed changes in cell size, shape, and septa formation, supporting the view that the phenomenon of adaptive resistance is operative in these isolates.

Differential transcription of multiple copies of a silk worm gene encoding tRNA

Ten different tRNAGly1 genes from the silk worm, Bombyx mori, have been cloned and characterized. These genes were transcribed in vitro in homologous nuclear extracts from the posterior silk gland (PSG) or nuclear extracts derived from the middle silk gland or ovarian tissues. Although the transcription levels were much higher in the PSG nuclear extracts, the transcriptional efficiency of the individual genes followed a similar pattern in all the extracts. Based on the levels of in vitro transcription, the ten tRNAGly1 genes could be divided into three groups, viz., those which were transcribed at very high levels (e.g., clone pR8), high to medium levels (e.g., pBmil, pBmpl, pBmhl, pBmtl) and low to barely detectable levels (e.g., pBmsl, pBmjl and pBmkl). The coding sequences of all these tRNA genes being identical, the differential transcription suggested that the flanking sequences modulate their transcriptional efficiency. The presence of positive and negative regulatory elements in the 5' flanking regions of these genes was confirmed by transcription competition experiments. A positive element was present in the immediate upstream A + T-rich sequences in all the genes, but no consensus sequences correlating to the transcriptional status could be generated. The presence of negative elements on the other hand was indicated only in some of the genes and therefore may have a role in the differential transcription of these tRNAGly genes in vivo.

Role of TATATAA element in the regulation of tRNA(1)(Gly) gene expression in Bombyx mori is position dependent

Transcription of tRNA genes by RNA polymerase III is controlled by the internal conserved sequences within the coding region and the immediate upstream flanking sequences. A highly transcribed copy of glycyl tRNA gene tRNA1(Gly)-1 from Bombyx mori is down regulated by sequences located much farther upstream in the region -150 to -300 nucleotides (nt), with respect to the +1 nt of tRNA. The negative regulatory effect has been narrowed down to a sequence motif 'TATATAA', a perfect consensus recognised by the TATA binding protein, TBP. This sequence element, when brought closer to the transcription start point, on the other hand, exerts a positive effect by promoting transcription of the gene devoid of other cis regulatory elements. The identity of the nuclear protein interacting with this 'TATATAA' element to TBP has been established by antibody and mutagenesis studies. The 'TATATAA' element thus influences the transcription of tRNA genes positively or negatively in a position-dependent manner either by recruitment or sequestration of TBP from the transcription machinery.

Effects Of 5' Flanking Sequences And Changes In The 5' Internal Control Region On The Transcription Of Rice Transfer-Rna Gcc Cly Gene

A stretch of 71 nucleotides in a 1.2 kilobase pair Pst I fragment of rice DNA was identified as tRNA~ gene by hybridization and nucleotide sequence analyses. The hybridization of genomic DNA with the tRNA gene showed that there are about 10 glycine tRNA genes per diploid rice genome. The 3' and 5' internal control regions, where RNA polymerase III and transcription factors bind, were found to be present in the coding sequence. The gene was transcribed into a 4S product in an yeast cell-free extract. The substitution of 5' internal control region with analogous sequences from either M13mpl9 or M13mpl8 DNA did not affect the transcription of the gene in vitro. The changes in three highly conserved nucleotides in the consensus 5' internal control region (RGYNNARYGG; R = purine, Y = pyrimidine, N = any nucleotide) did not affect transcription showing that these nucleotides are not essential for promotion of transcription. There were two 16 base pair repeats, 'TGTTTGTTTCAGCTTA' at - 130 and - 375 positions upstream from the start of the gene. Deletion of 5' flanking sequences including the 16 base pair repeat at - 375 showed increased transcription indicating that these sequences negatively modulate the expression of the gene.

Positional Preferences of Polypurine/Polypyrimidine Tracts in Saccharomyces cerevisiae Genome: Implications for cis Regulation of Gene Expression

The complete genome of the baker's yeast S. cerevisiae was analyzed for the presence of polypurine/polypyrimidine (poly[pu/py]) repeats and their occurrences were classified on the basis of their location within and outside open reading frames (ORFs). The analysis reveals that such sequence motifs are present abundantly both in coding as well as noncoding regions. Clear positional preferences are seen when these tracts occur in noncoding regions. These motifs appear to occur predominantly at a unit nucleosomal length both upstream and downstream of ORFs. Moreover, there is a biased distribution of polypurines in the coding strands when these motifs occur within open reading frames. The significance of the biased distribution is discussed with reference to the occurrence of these motifs in other known mRNA sequences and expressed sequence tags. A model for cis regulation of gene expression is proposed based on the ability of these motifs to form an intermolecular triple helix structure when present within the coding region and/or to modulate nucleosome positioning via enhanced histone affinity when present outside coding regions.

Transcriptional Silencing of a tRNA1Gly Copy from within a Multigene Family Is Modulated by Distal cis Elements

Individual copies of tRNA1Gly from within the multigene family in Bombyx mori could be classified based on in vitro transcription in homologous nuclear extracts into three categories of highly, moderately, or weakly transcribed genes. Segregation of the poorly transcribed gene copies 6 and 7, which are clustered in tandem within 425 base pairs, resulted in enhancement of their individual transcription levels, but the linkage itself had little influence on the transcriptional status. For these gene copies, when fused together generating a single coding region, transcription was barely detectable, which suggested the presence of negatively regulating elements located in the far flanking sequences. They exerted the silencing effect on transcription overriding the activity of positive regulatory elements. Systematic analysis of deletion, chimeric, and mutant constructs revealed the presence of a sequence element TATATAA located beyond 800 nucleotides upstream to the coding region acting as negative modulator, which when mutated resulted in high level transcription. Conversely, a TATATAA motif reintroduced at either far upstream or far downstream flanking regions exerted a negative effect on transcription. The location of cis-regulatory sequences at such farther distances from the coding region and the behavior of TATATAA element as negative regulator reported here are novel. These element(s) could play significant roles in activation or silencing of genes from within a multigene family, by recruitment or sequestration of transcription factors.

Nucleosomes on linear duplex DNA allow homologous pairing but prevent strand exchange promoted by RecA protein

To understand the molecular basis of gene targeting, we have studied interactions of nucleoprotein filaments comprised of single-stranded DNA and RecA protein with chromatin templates reconstituted from linear duplex DNA and histones. We observed that for the chromatin templates with histone/DNA mass ratios of 0.8 and 1.6, the efficiency of homologous pairing was indistinguishable from that of naked duplex DNA but strand exchange was repressed. In contrast, the chromatin templates with a histone/DNA mass ratio of 9.0 supported neither homologous pairing nor strand exchange. The addition of histone H1, in stoichiometric amounts, to chromatin templates quells homologous pairing. The pairing of chromatin templates with nucleoprotein filaments of RecA protein-single-stranded DNA proceeded without the production of detectable networks of DNA, suggesting that coaggregates are unlikely to be the intermediates in homologous pairing. The application of these observations to strategies for gene targeting and their implications for models of genetic recombination are discussed.

RAD51C: a novel cancer susceptibility gene is linked to Fanconi anemia and breast cancer

Germline mutations in many of the genes that are involved in homologous recombination (HR)-mediated DNA double-strand break repair (DSBR) are associated with various human genetic disorders and cancer. RAD51 and RAD51 paralogs are important for HR and in the maintenance of genome stability. Despite the identification of five RAD51 paralogs over a decade ago, the molecular mechanism(s) by which RAD51 paralogs regulate HR and genome maintenance remains obscure. In addition to the known roles of RAD51C in early and late stages of HR, it also contributes to activation of the checkpoint kinase CHK2. One recent study identifies biallelic mutation in RAD51C leading to Fanconi anemia-like disorder. Whereas a second study reports monoallelic mutation in RAD51C associated with increased risk of breast and ovarian cancer. These reports show RAD51C is a cancer susceptibility gene. In this review, we focus on describing the functions of RAD51C in HR, DNA damage signaling and as a tumor suppressor with an emphasis on the new roles of RAD51C unveiled by these reports.

Cloning and characterization of a cluster of genes coding for 5S rRNA and three tRNAs from Azospirillum lipoferum

A genomic library was constructed from a HindIII digest of Azospirillum lipoferum chromosomal DNA in the HindIII site of pUC19. From the library, a clone, pALH64, which showed strong hybridization with 3' end labeled A. lipoferum total tRNAs and which contains a 2.9 kb insert was isolated and restriction map of the insert established. The nucleotide sequence of a 490 bp HindIII-HincII subfragment containing a cluster of genes coding for 5S rRNA, tRNA(Val)(UAC), tRNA(Thr)(UGA) and tRNA(Lys)(UUU) has been determined. The gene organization is 5S rRNA (115 bp), spacer (10 bp), tRNA(Val) (76 bp), spacer (3 bp), tRNA(Thr) (76 bp), spacer (7 bp) and tRNA(Lys) (76 bp). Hybridization experiments using A. lipoferum total tRNAs and 5S rRNA with the cloned DNA probes revealed that all three tRNA genes and the 5S rRNA gene are expressed in vivo in the bacterial cells.

Characterization of the gene encoding the envelope fusion glycoprotein GP64 from Bombyx mori nucleopolyhedrovirus

We describe here the characterization of the gene gp64 encoding the envelope fusion protein GP64 (open reading frame) ORF 105 from Bombyx mori nucleopolyhedrovirus (BmNPV). gp64 was transcribed from the early to late stages of infection and the transcripts were seen from 6 to 72 h post infection (hpi). The early transcripts initiated from a consensus CAGT motif while the late transcripts arose from three conserved TAAG motifs, all of which were located in the near upstream region of the coding sequence. Both early and late transcripts terminated at a run of T residues following the second polyadenylation signal located 31 nt downstream of the translation termination codon. BmGP64 protein was detectable from 6 hpi and was present in larger quantities throughout the infection process from 12 hpi, in BmNPV-infected BmN cells. The persistent presence of GP64 in BmN cells differed from the protein expression pattern of GP64 in Autographa californica multinucleocapsid nucleopolyhedrovirus infection, where the protein levels decreased significantly by late times (48 hpi). BmGP64 was located in the membrane and cytoplasm of the infected host cells and as a component of the budded virions. The production of infectious budded virus and the fusion activity were reduced when glycosylation of GP64 was inhibited. (C) 2003 Elsevier Science B.V. All rights reserved.

Identification of a core promoter and a novel isoform of the human TSCI gene transcript and structural comparison with mouse homolog

Tuberous sclerosis complex (TSC) is an autosomal dominant disorder with loci on chromosome 9q34.12 (TSC1) and chromosome 16p13.3 (TSC2). Genes for both loci have been isolated and characterized. The promoters of both genes have not been characterized so far and little is known about the regulation of these genes. This study reports the characterization of the human TSC1 promoter region for the first time. We have identified a novel alternative isoform in the 5' untranslated region (UTR) of the TSC1 gene transcript involving exon 1. Alternative isoforms in the 5' UTR of the mouse Tsc1 gene transcript involving exon I and exon 2 have also been identified. We have identified three upstream open reading frames (uORFs) in the 5' UTR of the TSC1/Tsc1 gene. A comparative study of the 5' UTR of TSC1/Tsc1 gene has revealed that there is a high degree of similarity not only in the sequence but also in the splicing pattern of both human and mouse TSC1 genes. We have used PCR methodology to isolate approximately 1.6 kb genomic DNA 5' to the TSC1 cDNA. This sequence has directed a high level of expression of luciferase activity in both HeLa and HepG2 cells. Successive 5' and 3' deletion analysis has suggested that a -587 bp region, from position +77 to -510 from the transcription start site (TSS), contains the promoter activity. Interestingly, this region contains no consensus TATA box or CAAT box. However, a 521-bp fragment surrounding the TSS exhibits the characteristics of a CpG island which overlaps with the promoter region. The identification of the TSC1 promoter region will help in designing a suitable strategy to identify mutations in this region in patients who do not show any mutations in the coding regions. It will also help to study the regulation of the TSC1 gene and its role in tumorigenesis. (C) 2003 Elsevier B.V. All rights reserved.

Development of a New Generation of Vectors for Gene Expression, Gene Replacement, and Protein-Protein Interaction Studies in Mycobacteria

Escherichia coli-mycobacterium shuttle vectors are important tools for gene expression and gene replacement in mycobacteria. However, most of the currently available vectors are limited in their use because of the lack of extended multiple cloning sites (MCSs) and convenience of appending an epitope tag(s) to the cloned open reading frames (ORFs). Here we report a new series of vectors that allow for the constitutive and regulatable expression of proteins, appended with peptide tag sequences at their N and C termini, respectively. The applicability of these vectors is demonstrated by the constitutive and induced expression of the Mycobacterium tuberculosis pknK gene, coding for protein kinase K, a serine-threonine protein kinase. Furthermore, a suicide plasmid with expanded MCS for creating gene replacements, a plasmid for chromosomal integrations at the commonly used L5 attB site, and a hypoxia-responsive vector, for expression of a gene(s) under hypoxic conditions that mimic latency, have also been created. Additionally, we have created a vector for the coexpression of two proteins controlled by two independent promoters, with each protein being in fusion with a different tag. The shuttle vectors developed in the present study are excellent tools for the analysis of gene function in mycobacteria and are a valuable addition to the existing repertoire of vectors for mycobacterial research.

Comparative genomics reveals `novel' Fur regulated sRNAs and coding genes in diverse proteobacteria

Ferric uptake regulator (Fur) is a transcriptional regulator controlling the expression of genes involved in iron homeostasis and plays an important role in pathogenesis. Fur-regulated sRNAs/CDSs were found to have upstream Fur Binding Sites (FBS). We have constructed a Positional Weight Matrix from 100 known FBS (19 nt) and tracked the `Orphan' FBSs. Possible Fur regulated sRNAs and CDSs were identified by comparing their genomic locations with the `Orphan' FBSs identified. Thirty-eight `novel' and all known Fur regulated sRNAs in nine proteobacteria were identified. In addition, we identified high scoring FBSs in the promoter regions of the 304 CDSs and 68 of them were involved in siderophore biosynthesis, iron-transporters, two-component system, starch/sugar metabolism, sulphur/methane metabolism, etc. The present study shows that the Fur regulator controls the expression of genes involved in diverse metabolic activities and it is not limited to iron metabolism alone. (C) 2012 Elsevier B.V. All rights reserved.

Mutation analysis of the SLC4A11 gene in Indian families with congenital hereditary endothelial dystrophy 2 and a review of the literature

Purpose: Congenital hereditary endothelial dystrophy 2 (CHED2) is an autosomal recessive disorder caused by mutations in the solute carrier family 4, sodium borate transporter, member 11 (SLC4A11) gene. The purpose of this study was to identify the genetic cause of CHED2 in six Indian families and catalog all known mutations in the SLC4A11 gene. Methods: Peripheral blood samples were collected from individuals of the families with CHED2 and used in genomic DNA isolation. PCR primers were used to amplify the entire coding region including intron-exon junctions of SLC4A11. Amplicons were subsequently sequenced to identify the mutations. Results: DNA sequence analysis of the six families identified four novel (viz., p.Thr262Ile, p.Gly417Arg, p.Cys611Arg, and p.His724Asp) mutations and one known p.Arg869His homozygous mutation in the SLC4A11 gene. The mutation p.Gly417Arg was identified in two families. Conclusions: This study increases the mutation spectrum of the SLC4A11 gene. A review of the literature showed that the total number of mutations in the SLC4A11 gene described to date is 78. Most of the mutations are missense, followed by insertions-deletions. The present study will be helpful in genetic diagnosis of the families reported here.

A Novel Mutation in the Transglutaminase-1 Gene in an Autosomal Recessive Congenital Ichthyosis Patient

Structure-function implication on a novel homozygous Trp250/Gly mutation of transglutaminase-1 (TGM1) observed in a patient of autosomal recessive congenital ichthyosis is invoked from a bioinformatics analysis. Structural consequences of this mutation are hypothesized in comparison to homologous enzyme human factor XIIIA accepted as valid in similar structural analysis and are projected as guidelines for future studies at an experimental level on TGM1 thus mutated.