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PubMed1983

Identification of repressor binding sites controlling expression of tetracycline resistance encoded by Tn10.

The regulatory region controlling the expression of tetracycline resistance and repressor genes contains two nearly identical regions of dyad symmetry. Deletions of this control region were isolated by digestion with S1 nuclease. The ability of these deletions to bind the tet repressor was determined by an in vivo repressor titration assay. The results indicate that repressor specifically binds both regions of dyad symmetry.

Base Sequence↗
PubMed1992

Identification of a repetitive element in the snail Biomphalaria glabrata: relationship to the reverse transcriptase-encoding sequence in LINE-1 transposons.

BamHI-digested Biomphalaria glabrata DNA contains a repetitive 2.0-kb fragment which is readily discernible by ethidium bromide staining. We present evidence that this repetitive element is related at both the nucleotide and amino acid levels to long interspersed nuclear element (LINE)-like transposons. Although comparable elements have been described in several invertebrates, this is the first report of a molluscan homologue. In common with LINE transposons, an open reading frame in the B. glabrata element shows significant homology to reverse transcriptase--a feature believed to allow the dissemination of these elements in the eukaryotic genome.

Amino Acid Sequence↗
PubMed1995

Molecular cloning and characterization of the dpy-20 gene of Caenorhabditis elegans.

We describe the molecular analysis of the dpy-20 gene in Caenorhabditis elegans. Isolation of genomic sequences was facilitated by the availability of a mutation that resulted from insertion of a Tc1 transposable element into the dpy-20 gene. The Tc1 insertion site in the m474::Tc1 allele was identified and was found to lie within the coding region of dpy-20. Three revertants (two wild-type and one partial revertant) resulted from the excision of this Tc1 element. Genomic dpy-20 clones' were isolated from a library of wild-type DNA and were found to lie just to the left of the unc-22 locus on the physical map, compatible with the position of dpy-20 on the genetic map. Cosmid DNA containing the dpy-20 gene was successfully used to rescue the mutant phenotype of animals homozygous for another dpy-20 allele, e1282ts. Sequence analysis of the putative dpy-20 homologue in Caenorhabditis briggsae was performed to confirm identification of the coding regions of the C. elegans gene and to identify conserved regulatory regions. Sequence analysis of dpy-20 revealed that it was not similar to other genes encoding known cuticle components such as collagen or cuticulin. The dpy-20 gene product, therefore, identifies a previously unknown type of protein that may be directly or indirectly involved in cuticle function. Northern blot analysis showed that dpy-20 is expressed predominantly in the second larval stage and that the mRNA is not at all abundant. Data from temperature shift studies using the temperature-sensitive allele e1282ts showed that the sensitive period also occurs at approximately the second larval stage. Therefore, expression of dpy-20 mRNA and function of the DPY-20 protein are closely linked temporally.

Amino Acid Sequence↗
PubMed2003

Genomics of the human Y-chromosome. 1. Association with male infertility.

The human Y chromosome contains over 60 million nucleotides, but least number of genes compared to any other chromosome and acts as a genetic determinant of the male characteristic features. The male specific region, MSY, comprising 95% of the Y chromosome represents a mosaic of heterochromatic and three classes of euchromatic (X-transposed, X-degenerate and ampliconic) sequences. Thus far, 156 transcription units, 78 protein-coding genes and 27 distinct proteins of the Y chromosome have been identified. The MSY euchromatic sequences show frequent gene conversion. Of the eight massive palindromes identified on the human Y chromosome, six harbor vital testis specific genes. The human male infertility has been attributed to mutations in the genes on Y chromosome and autosomes and failures of several physical and physiological attributes including paracrine controls. In addition, deletion of any one or all the three azoospermia (AZFa, AZFb or AZFc) factor(s) and some still unidentified regulatory elements located elsewhere in the genome result in infertility. Characterization of palindromic complexes on the long arm of Y chromosome encompassing AZFb and AZFc regions and identification of HERV15 class of endogenous retroviruses close to AZFa region have facilitated our understanding on the organization of azoospermia factors. Considerable overlap of the AZFb and AZFc regions encompassing a number of genes and transcripts has been shown to exist. However, barring details on AZF, information on the exact number of genes or the types of mutations prevalent in the infertile male is not available. Similarly, roles of sizable body of repetitive DNA present in close association with transcribing sequences on the Y chromosome are yet not clear. In a clinical setting with known cases of infertility, systematic search for loss or gain of these repeat elements would help understand their biological role(s). We present a brief overview on the genetic complexity of the human Y chromosome in the context of human male infertility.

Chromosomes, Human, Y↗
PubMed2025

LncRAnalyzer: a robust workflow for long non-coding RNA discovery using RNA-Seq.

Long non-coding RNA (lncRNA) is a major transcript category that lacks protein-coding capabilities, with relatively low abundance and complex expression patterns. Distinguishing lncRNAs from protein-coding genes is a complex process involving multiple filtering steps. We developed an automated pipeline named LncRAnalyzer featuring retrained models for 60 species. This workflow aims to reduce the likelihood of obtaining protein-coding or partial protein-coding transcripts during lncRNA identification by utilizing eight distinct approaches. We conducted a 10-fold cross-validation of the sorghum models and training sets with their standard ones and other approaches using real-life RNA-Seq datasets and known lncRNA and CDS sequences of sorghum. The results showed that the sorghum models and training sets were outperformed. The pipeline output comprises upset plots illustrating the number of lncRNA/NPCTs identified by the approaches, commonly identified lncRNA and their classes, NPCTs, and expression count tables. A feature-level comparison and benchmarking analysis of LncRAnalyzer with four existing pipelines, namely, LncPipe, LncEvo, lncRNA-Annotation, and Plant-LncPipe, demonstrated that LncRAnalyzer is more comprehensive, easier to implement, and accurate in lncRNA predictions. This workflow also ascertains lncRNA origins from various Transposable Elements (TEs) in plants using TE annotations from APTEdb [http://apte.cp.utfpr.edu.br/]. LncRAnalyzer is publicly available on GitLab [https://gitlab.com/nikhilshinde0909/LncRAnalyzer.git] for academic users.

RNA, Long Noncoding↗
PubMed1998

A shuttle mutagenesis system for tagging genes in the yeast Yarrowia lipolytica.

A shuttle mutagenesis system was developed for the dimorphic yeast Yarrowia lipolytica. This system combines transposon insertions generated in Escherichia coli with the transformation of yeast with the Tn-mutagenized DNA. The mini-transposon mTn-3xHA/GFP, used in Saccharomyces cerevisiae for producing stable insertions, was adapted for use in the yeast Y. lipolytica. The mTnYl1 transposon (for mini-Tn of Y. lipolytica) confers resistance to tetracycline in E. coli. It also contains the Y. lipolytica URA3 gene for selection of yeast transformants, and the coding sequence for the S65T mutant form of GFP. The rare cutter endonuclease, I-SceI, restriction site, which enables identification of the chromosomal localization of mutagenized genes, was also incorporated. mTnYl1 was first tested on the ACO1 gene, which encodes an Acyl CoA oxidase isozyme. The mutagenesis system was further validated on a Y. lipolytica genomic DNA library constructed in a pHSS6 derivative vector. Mutants with a particular morphology or defective for alkane, fatty acids and oil degradation were obtained.

Acyl-CoA Oxidase↗
PubMed2003

In silico identification of putative regulatory sequence elements in the 5'-untranslated region of genes that are expressed during male gametogenesis.

During pollen development, transcription of a large number of genes results in the appearance of distinct sets of transcripts. Similar mRNA sets are present in pollen of both mono- and dicotyledonous plant species, which indicates an evolutionary conservation of genetic programs that determine pollen gene expression. In pollen, regulation of gene expression occurs at the transcriptional and posttranscriptional level. The 5'-untranslated region (UTR) of several pollen transcripts has been shown to be important for regulation of pollen gene expression. The important regulatory role of 5'-UTR sequences and the evolutionary conservation of genetic programs in pollen led to the hypothesis that the 5'-UTRs of pollen-expressed genes share regulatory sequence elements. In an attempt to identify these pollen 5'-UTR elements, a statistical analysis was performed using 5'-UTR sequences of pollen- and sporophytic-expressed genes. The analysis revealed the presence of several pollen-specific 5'-UTR sequence elements. Assembly of the pollen 5'-UTR elements led to the identification of various consensus sequences, including those that previously have been demonstrated to play a role in the regulation of pollen gene expression. Several pollen 5'-UTR elements were found to be preferentially associated to genes from dicots, wet-type stigma plants, or plants containing bicellular pollen. Moreover, three sequence elements exhibited a preferential association to the 5'-UTR of pollen-expressed genes from Arabidopsis and Brassica napus. Functional implications of these observations are discussed.

5' Untranslated Regions↗
PubMedDate not supplied

Insertion-deletion polymorphisms in 3' regions of maize genes occur frequently and can be used as highly informative genetic markers.

Single-nucleotide polymorphisms (SNPs) are the most frequent variations in the genome of any organism. SNP discovery approaches such as resequencing or data mining enable the identification of insertion deletion (indel) polymorphisms. These indels can be treated as biallelic markers and can be utilized for genetic mapping and diagnostics. In this study 655 indels have been identified by resequencing 502 maize (Zea mays) loci across 8 maize inbreds (selected for their high allelic variation). Of these 502 loci, 433 were polymorphic, with indels identified in 215 loci. Of the 655 indels identified, single-nucleotide indels accounted for more than half (54.8%) followed by two- and three-nucleotide indels. A high frequency of 6-base (3.4%) and 8-base (2.3%) indels were also observed. When analysis is restricted to the B73 and Mol7 genotypes, 53% of the loci analyzed contained indels, with 42% having an amplicon size difference. Three novel miniature inverted-repeat transposable element (MITE)-like sequences were identified as insertions near genes. The utility of indels as genetic markers was demonstrated by using indel polymorphisms to map 22 loci in a B73 x Mo17 recombinant inbred population. This paper clearly demonstrates that the resequencing of 3' EST sequence and the discovery and mapping of indel markers will position corresponding expressed genes on the genetic map.

Chromosome Mapping↗
PubMed1994

Shuttle mutagenesis of Legionella pneumophila: identification of a gene associated with host cell cytopathicity.

We performed shuttle mutagenesis of Legionella pneumophila. Mutants were screened for reduced cellular infectivity. Approximately 10% of the mutants had decreased cytopathicity. The DNA sequence of one locus was determined; the inferred amino acid sequence revealed homology with transport proteins including Escherichia coli TolC, Bordetella pertussis CyaE, and Alcaligenes eutrophus CzcC and CnrC.

Amino Acid Sequence↗
PubMed1993

Copia-like retrotransposable element evolution in diploid and polyploid cotton (Gossypium L.).

Copia-like retrotransposable elements were identified in allotetraploid cotton, Gossypium hirsutum, and two species representing its diploid progenitors, G. herbaceum and G. raimondii. These elements are present in high copy number in all three species. Because the two diploid genomic groups have been isolated on opposite sides of the world for 6-11 million years, horizontal transfer of elements between these species is highly unlikely. Elements were intensively sampled to generate a model of copia-like retrotransposable element evolution in systems where vertical transmission is the sole probable means of descent. Copia-like retrotransposon diversity is equally great in all three Gossypium species. Despite this high heterogeneity, analysis of 89 partial reverse transcriptase sequences resulted in the recognition of nine sharply differentiated retrotransposon lineages, each containing elements that share high sequence similarity. No evidence of horizontal transfer from other taxa was obtained. Phylogenetic analyses demonstrate that element topologies are incongruent with Gossypium phylogeny. Consideration of processes that obscure phylogenetic reconstruction of multigene families (including sampling error, variable degrees of orthology and paralogy, differential lineage age and lineage loss and/or proliferation) demonstrates that incongruence between organismal and retrotransposon trees is expected under conditions in which vertical processes are the sole means of transmission. Identification of closely related elements between species allowed rates of copia-like retrotransposon sequence evolution to be estimated as approximately 10(-9) nucleotide substitutions/site/year. These rates are consistent with the interpretation that these retrotransposons have been evolving under functional constraints for most of the time frame bracketed by the species studied. Extrapolation of these results to previous studies that sampled from more highly divergent taxa indicates that horizontal transfer need not be invoked to explain observed phylogenetic patterns.

Amino Acid Sequence↗
PubMed1991

Identification of an insertion sequence, IS1081, in Mycobacterium bovis.

An insertion sequence, IS1081, in the genome of Mycobacterium bovis has been identified and sequenced. It is 1324 bp long with 15 bp inverted repeat ends and contains a large ORF. There are six copies of IS1081 in the genome of M. bovis and the element is also present in Mycobacterium tuberculosis. IS1081 is not closely related to other DNA elements described in actinomycetes but its putative transposase bears some resemblance to that of IS256 from Staphylococcus aureus. IS1081 may be useful for genetic manipulations and for developing a diagnostic test for bovine tuberculosis based on the polymerase chain reaction.

Base Sequence↗
PubMed1996

Diversification of C-function activity in maize flower development.

The Arabidopsis gene AGAMOUS is required for male and female reproductive organ development and for floral determinacy. Reverse genetics allowed the isolation of a transposon-induced mutation in ZAG1, the maize homolog of AGAMOUS. ZAG1 mutants exhibited a loss of determinacy, but the identity of reproductive organs was largely unaffected. This suggested a redundancy in maize sex organ specification that led to the identification and cloning of a second AGAMOUS homolog, ZMM2, that has a pattern of expression distinct from that of ZAG1. C-function organ identity in maize (as defined by the A, B, C model of floral organ development) may therefore be orchestrated by two closely related genes, ZAG1 and ZMM2, with overlapping but nonidentical activities.

Alleles↗
PubMed1993

The Bradyrhizobium japonicum serocluster 123 hyperreiterated DNA region, HRS1, has DNA and amino acid sequence homology to IS1380, an insertion sequence from Acetobacter pasteurianus.

We have sequenced and analyzed the hyperreiterated DNA region, HRS1, from Bradyrhizobium japonicum USDA 424. The 2.1-kb HRS1 fragment is closely linked to the B. japonicum common and genotype-specific nodulation genes in serogroup 123 and 127 strains. Southern hybridization analyses indicated that one copy of HRS1 is also located next to the fixRnifA locus in B. japonicum USDA 424. Nucleotide sequence analysis revealed the presence of a 4-bp target site duplication in HRS1 which is identical to a terminal repeat found in the B. japonicum USDA 110 repeated sequence RS alpha. Computer searches of the PIR (Protein Identification Resource) protein data base revealed a high degree of amino acid sequence homology between a putative 329-amino-acid polypeptide from HRS1 and a large polypeptide from IS1380, an insertion sequence from Acetobacter pasteurianus. RNA slot blot hybridizations suggest that transcripts showing homology to HRS1 are constitutively produced in strains USDA 424 (serogroup 127) and USDA 438 (serogroup 123).

Acetobacter↗
PubMed1991

Tec2, a second transposon-like element demonstrating developmentally programmed excision in Euplotes crassus.

The analysis of a repetitive DNA interruption of the micronuclear precursor to a 0.85-kb macronuclear gene in the hypotrich Euplotes crassus has led to the identification of a second transposon-like element named Tec2. Two copies of this element, one inserted into the other, compose the interruption. The Tec2 element resembles the previously characterized Tec1 element in overall size, copy number, length, and extreme terminal sequence of its inverted repeats and in the apparent use of a 5'-TA-3' target site. In addition, extrachromosomal circular forms of Tec2 appear in DNA isolated from cells undergoing macronuclear development at the same time and with the same conformation as extrachromosomal circular forms of Tec1. These similarities suggest that the Tec1 and Tec2 elements may be under the same type of regulation during macronuclear development.

Animals↗
PubMed2005

Identification of insertion sequence from a gamma-hexachlorocyclohexane degrading bacterium, Sphingomonas paucimobilis UT26.

Tn5-derived mutants of the gamma-hexachlorocyclohexane-degrading bacterium Sphingomonas paucimobilis UT26 were genetically characterized, and an endogenous insertion sequence (IS) which belongs to the IS1380 family was identified. The IS, named ISsp1, existed as multi copies in UT26, and its transposition appeared to be activated during the process of Tn5-mutagenesis. It was found that transposon mutagenesis can cause endogenous mutations.

DNA Transposable Elements↗
PubMed1996

Identification of Porto-1, a new repeated sequence that localises close to the centromere of chromosome 2 of Drosophila melanogaster.

We have used the polymerase chain reaction (PCR) technique to search the Drosophila melanogaster genome for the presence of sequences with homology to mammalian and yeast centromeric DNA. Using primers based on the human CENP-B box present in alpha-satellite DNA and part of the Saccharomyces cerevisiae CDEIII centromeric sequence, a number of specific DNA fragments were amplified from total genomic DNA. In situ hybridization to polytene and mitotic chromosomes showed these fragments to localise to centromeric and pericentromeric regions. Direct cloning of the amplified fragments into conventional plasmids proved unsuccessful. However, a recombinant P1 clone containing D. melanogaster genomic DNA that supports PCR amplification by the primers was identified. Molecular characterisation of this clone revealed a DNA fragment that localises primarily to the centromere of chromosome 2. Sequence analysis indicated that this fragment contains at least four different repeats, including Rsp, transposable elements, Bari-1 and a new AT-rich repeated sequence that we have designated Porto-1. Detailed fluorescence in situ hybridization analysis shows that Porto-1 is localised very close to the primary constriction of chromosome 2. Sequence analysis suggests that this repeat was specifically amplified by our primers, although limited homology to the CENP-B box or CDEIII elements was found. In situ hybridization to a number of Drosophila species shows Porto-1 to be present only in D. melanogaster.

Amino Acid Sequence↗
PubMed1995

Identification of a mariner element from the tsetse fly, Glossina palpalis palpalis.

In the present study, the polymerase chain reaction was used initially to demonstrate the presence of mariner sequences in seven species/subspecies of tsetse flies. DNA hybridization experiments show mariner sequences to be dispersed within the tsetse genome and that there are large variations in copy numbers among the various taxa. A genomic library was used to isolate and characterize a full-length mariner element from G. p. palpalis. The results indicate that this element is 1257 bp in length, flanked by two 32 bp inverted repeats differing at only one position, and belongs to the mellifera subfamily. The nucleotide sequence that is translated into a reading frame of 337 amino acids requires the introduction of two frame shifts and one stop codon to maximize sequence homology with a mariner element from Drosophila erecta. Based on this evidence, we conclude that the G. p. palpalis mariner element clearly represents a non-functional transposable element and that the protein product is not an active transposase.

Amino Acid Sequence↗