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PubMed2006

Competition may determine the diversity of transposable elements.

Transposable elements are genomic parasites that replicate independently from their hosts. They harm their hosts by causing mutations or genomic rearrangements, and most organisms have evolved various mechanisms to suppress their activity. The evolutionary dynamics of transposons in insects, fish, birds and mammals are dramatically different. Mammalian genomes contain few, very abundant but relatively inactive transposon strains, while Drosophila and fish species harbour diverse strains, which typically have low abundance but are much more virulent. We hypothesise that the variation in the diversity and activity of transposable elements between various animal genomes is caused by the differences in the host defence mechanisms against transposon activity. In recent years RNAi, a mechanism capable of gene, virus and transposon silencing has been discovered. We model RNAi as a density dependant mechanism of defence, which can cause competition among transposons depending on its specificity, and test its predictions using the complete Caenorhabditis elegans, Drosophila melanogaster, Fugu rubripes, chicken, mouse, rat and human genome sequences.

Animals↗
PubMed1997

Analysis of single-strand conformation polymorphisms by capillary electrophoresis with laser induced fluorescence detection.

Detection of point mutations in genomic DNA is important for diagnosis of inherited characteristics and genetic diseases. A point mutation in a specific region of DNA amplified by polymerase chain reaction (PCR) can be detected with single-strand conformation polymorphism (SSCP) analysis. Analysis of SSCP by laser-induced fluorescence capillary electrophoresis in entangled polymer solution (CE-LIF) has been developed in the present paper. K-ras genes including seven mutations were amplified with primer labeled with Texas Red at its 5' end. The labeled PCR products were dissociated to single strands by heating and separated with capillary gel electrophoresis and He-Ne laser-excited fluorescence detection. Our results suggest that all fragments having normal (Gly) and mutated (Ala, Arg, Cys, Ser, Val, Asp) sequences at codon 12 can be distinguished. Analysis of SSCPs with CE-LIF is well suited for clinical analysis of SSCPs because of its high sensitivity, resolution, reproducibility and speed.

Codon↗
PubMed1986

Mapping and sequencing of RNAs without recourse to molecular cloning: application to RNAs of the Sabin 1 strain of poliovirus and its defective interfering particles.

Complementary DNA to the genome of the Sabin 1 strain of poliovirus was prepared by reverse transcription with oligo(dT)10 as a primer and separated into six classes of DNA by their size. Each class of the DNA, after digestion with restriction endonuclease HaeIII, was analyzed by two-dimensional polyacrylamide gel electrophoresis. Comparison of the patterns of the restriction fragments led us to compose a possible arrangement of the restriction fragments on the viral genome. Sequence analysis of these fragments indicated that the arrangement was consistent with the known total nucleotide sequence of the genome. In the determined sequences, two bases were observed to differ from those of a cloned complementary DNA of the Sabin 1 genome. This suggested that the sequence of the cloned DNA reflected that of a mutated virus genome that was a minor component in the virus inoculation stock. The genomes of defective interfering particles generated from the Sabin 1 strain were also analyzed by this technique. The results suggested that the RNAs lacked an internal region of the Sabin 1 RNA encoding viral capsid proteins. The location of the deletion was further confirmed by determination of the nucleotide sequence of a cloned complementary DNA copy of the defective interfering particle RNA. Thus, the method described here is useful for mapping and sequencing of RNAs and for knowing whether cloned cDNAs represent the major population of RNA molecules or not.

Base Sequence↗
PubMed2001

Mutagenesis in the post genomics era: tools for generating insertional mutations in the lactic acid bacteria.

The increasing availability of whole genome sequences has increased the demand for effective tools to generate insertional mutations in the lactic acid bacteria (LAB). Several novel approaches, such as shuttle-, transposome- and intron-based mutagenesis methods, are possible additions to the existing repertoire of transposon- and recombination-based tools available for mutagenesis of LAB.

Bacteria↗
PubMed2005

G to A hypermutation of hepatitis B virus.

G to A hypermutation of the human immunodeficiency virus type 1 (HIV-1) is induced by a deaminase APOBEC3G and is related to host antiviral defense. APOBEC3G has also been found to reduce the replication of HIV-1 by an unknown mechanism. This enzyme also reduces the production of hepatitis B virus, although the mechanism for this action has not been clearly elucidated. The hypermutated hepatitis B virus (HBV) is rarely found in usual sequencing analyses. Using peptide nucleic acid mediated by polymerase chain reaction clamping, we detected the hypermutated HBV DNA in 1 of 8 patients with acute HBV infection and 4 of 10 with chronic HBV infection. In the latter group, hypermutated genomes were found only in eAb-positive patients. As much as 72.5% of G residues were mutated in the hypermutated clones. G to A substitutions were predominant in almost all clones sequenced compared with other substitutions. G to A mutated viral genomes also were found in HepG2-derived cell lines that continuously produced HBV into the supernatant. Both alpha and gamma interferon reduced virus production in these cell lines, but they did not alter the frequency of the hypermutation. Transcripts of APOBEC3G, as well as some other deaminases, were found in these cell lines. In conclusion, our results show that part of the minus strand DNA of HBV is hypermutated both in vitro (HepG2 cell lines) and in vivo. The role and mechanism of hypermutation in reducing HBV replication should be further investigated to understand the anti-HBV defense system.

APOBEC-3G Deaminase↗
PubMed1992

Attenuation of Sindbis virus neurovirulence by using defined mutations in nontranslated regions of the genome RNA.

We examined a panel of Sindbis virus mutants containing defined mutations in the 5' nontranslated region of the genome RNA, in the 3' nontranslated region, or in both for their growth in cultured cells and virulence in newborn mice. In cultured cells, these viruses all had defects in RNA synthesis and displayed a wide range of growth rates. The growth properties of the mutants were often very different in mouse cells from those in chicken cells or in mosquito cells. We hypothesize that host factors, presumably proteins, interact with these nontranslated regions to promote viral replication and that the mammalian protein and the chicken or mosquito protein are sufficiently divergent that alterations in the viral RNA sequence can affect the interactions with these different host proteins in different ways. Some of the mutants were temperature sensitive for plaque formation, whereas one mutant was slightly cold sensitive in its growth in chicken cells. Upon inoculation into mice, viruses that grew well in cultured mouse cells retained their virulence, but mice that succumbed usually had extended survival times. One virulent mutant that grew slightly less well in cultured mouse cells than did the parental virus produced eight times as much virus in mouse brain following intracerebral inoculation, suggesting that changes in these regulatory regions may have tissue-specific as well as host-specific effects. Viruses that were severely crippled in their growth in mouse cells in culture were usually, but not always, attenuated in their virulence. In particular, temperature sensitivity was correlated with attenuation. The effect of two mutations was found to be cumulative, and double mutants that contained mutations in both the 5' and 3' nontranslated regions were more attenuated than was either single mutant. Three of four double mutants tested were severely crippled for virus production in cultured cells and were avirulent for mice, even when inoculated intracerebrally.

Animals↗
PubMed1999

Primary and secondary structural elements required for synthesis of barley yellow dwarf virus subgenomic RNA1.

Barley yellow dwarf luteovirus (BYDV) generates three 3'-coterminal subgenomic RNAs (sgRNAs) in infected cells. The promoter of sgRNA1 is a putative hot spot for RNA recombination in luteovirus evolution. The sgRNA1 transcription start site was mapped previously to either nucleotide 2670 or nucleotide 2769 of BYDV genomic RNA (gRNA) in two independent studies. Our data support the former initiation site. The boundaries of the sgRNA1 promoter map between nucleotides 2595 and 2692 on genomic RNA. Computer prediction, phylogenetic comparison, and structural probing revealed two stem-loops (SL1 and SL2) in the sgRNA1 promoter region on the negative strand. Promoter function was analyzed by inoculating protoplasts with a full-length infectious clone of the BYDV genome containing mutations in the sgRNA promoter. Because the promoter is located in an essential coding region of the replicase gene, we duplicated it in a nonessential part of the genome from which a new sgRNA was expressed. Mutational analysis revealed that secondary structure, but not the nucleotide sequence, was important at the base of SL1. Regions with both RNA primary and secondary structural features that contributed to transcription initiation were found at the top of SL1. Primary sequence, but not the secondary structure, was required in SL2, which includes the initiation site. Disruption of base pairing near the sgRNA1 start site increased the level of transcription three- to fourfold. We propose that both primary and secondary structures of the sgRNA1 promoter of BYDV play unique roles in sgRNA1 promoter recognition and transcription initiation.

Base Sequence↗
PubMed1998

Peripheral blood mononuclear cells express mutated NCCT mRNA in Gitelman's syndrome: evidence for abnormal thiazide-sensitive NaCl cotransport.

Genetic analysis has demonstrated complete linkage between the human thiazide-sensitive sodium chloride cotransporter gene (NCCT or TSC) and Gitelman's syndrome (GS). Several genomic NCCT mutations have been reported. This study was performed to determine whether peripheral blood mononuclear cells (PBMC) express NCCT mRNA and whether defective PBMC NaCl cotransport could be demonstrated in GS. PBMC were isolated from two brothers with GS, their parents, and healthy control subjects. Northern analysis revealed that NCCT mRNA is expressed in PBMC. The sequence of full-length NCCT cDNA amplified from normal PBMC was identical to human renal NCCT cDNA. Two different mutations were detected in the patients' NCCT cDNA (compound heterozygote). In cDNA derived from the patient's maternal allele, exon 24 was deleted, resulting in a premature stop codon (after amino acid 920). cDNA derived from the patient's paternal allele had an additional 119-bp insertion between exons 3 and 4, generating a premature stop codon (after amino acid 187). The patient's genomic DNA had a previously described 5' splice site mutation in intron 24, GGT --> GTT (maternal allele), and a new 3' splice site mutation in intron 3, CAG --> CAA (paternal allele), which resulted in the activation of a nearby cryptic splice site in intron 3. The latter mutation was not present in 300 normal chromosomes. To determine the functional significance of these findings, chlorothiazide-inhibitable 22Na uptake was measured in PBMC from control subjects, the parents, and the patients with GS in the presence of bumetanide. In control PBMC, chlorothiazide inhibited 22Na uptake by approximately 9%. PBMC from the two patients with GS failed to respond to chlorothiazide. These results demonstrate that PBMC can be used for mutational analysis of NCCT mRNA in patients with GS. Furthermore, functional evidence is provided that the underlying cause of GS is defective NCCT NaCl cotransport.

Adult↗
PubMed2004

Site-specific genomic (SSG) and random domain-localized (RDL) mutagenesis in yeast.

BACKGROUND: A valuable weapon in the arsenal available to yeast geneticists is the ability to introduce specific mutations into yeast genome. In particular, methods have been developed to introduce deletions into the yeast genome using PCR fragments. These methods are highly efficient because they do not require cloning in plasmids. RESULTS: We have modified the existing method for introducing deletions in the yeast (S. cerevisiae) genome using PCR fragments in order to target point mutations to this genome. We describe two PCR-based methods for directing point mutations into the yeast genome such that the final product contains no other disruptions. In the first method, site-specific genomic (SSG) mutagenesis, a specific point mutation is targeted into the genome. In the second method, random domain-localized (RDL) mutagenesis, a mutation is introduced at random within a specific domain of a gene. Both methods require two sequential transformations, the first transformation integrates the URA3 marker into the targeted locus, and the second transformation replaces URA3 with a PCR fragment containing one or a few mutations. This PCR fragment is synthesized using a primer containing a mutation (SSG mutagenesis) or is synthesized by error-prone PCR (RDL mutagenesis). In SSG mutagenesis, mutations that are proximal to the URA3 site are incorporated at higher frequencies than distal mutations, however mutations can be introduced efficiently at distances of at least 500 bp from the URA3 insertion. In RDL mutagenesis, to ensure that incorporation of mutations occurs at approximately equal frequencies throughout the targeted region, this region is deleted at the same time URA3 is integrated. CONCLUSION: SSG and RDL mutagenesis allow point mutations to be easily and efficiently incorporated into the yeast genome without disrupting the native locus.

Genes, Fungal↗
PubMed1994

p53 gene mutations and steroid receptor status in breast cancer. Clinicopathologic correlations and prognostic assessment.

BACKGROUND: There is increasing evidence linking development and progression of cancer to an accumulation of mutations at the genomic level. The most frequently mutated gene known to date in sporadic breast cancer appears to be the tumor suppressor gene p53. This study was designed to determine the frequency of p53 gene mutations in primary breast cancer, to correlate the presence of p53 mutations with established clinicopathologic parameters, including the estrogen receptor (ER) and progesterone receptor (PR) status, and to assess the prognostic significance of p53 mutations regarding patient survival. METHODS: We examined the p53 gene in genomic DNA samples from 192 primary breast cancers. Using denaturant gradient gel electrophoresis, the authors analyzed exons 5-9 in all tumors for mutations and performed DNA sequencing in 20 tumors to identify the exact nature of the p53 mutations. RESULTS: p53 gene alterations were identified in 43 of the 192 tumors (22%), the majority localized in exons 5 and 6. DNA sequencing showed mostly missense mutations resulting from G or C substitutions. p53 mutations were found more often in tumors of younger women (P = 0.002), Afro-American women (P = 0.05), and in tumors lacking ER (P = 0.03), PR (P = 0.04), or both (P = 0.06). There were no significant correlations with family history, tumor size, histologic grade or type, nodal status, or disease stage. The overall survival rates showed no significant difference between patients with mutant and wild-type p53 tumors. The same was true when the comparison was limited to node-negative patients or patients with ER-positive or ER-negative tumors. Finally, there was no significant difference in survival between patients with tumors harboring mutations in exons 5 and 6 versus exons 7-9. CONCLUSIONS: The results of this and other studies demonstrate a consistent relationship between ER-positive tumors and wild-type p53 on one hand and ER-negative cancers and p53 mutations on the other. Our data do not support a significant prognostic role for p53 mutations in predicting survival.

Base Sequence↗
PubMed1994

Retrotransposon-induced ectopic expression of the Om(2D) gene causes the eye-specific Om(2D) phenotype in Drosophila ananassae.

Optic morphology (Om) mutations in Drosophila ananassae map to at least 22 loci, which are scattered throughout the genome. Om mutations are all semidominant, neomorphic, nonpleiotropic, and associated with the insertion of a retrotransposon, tom. We have found that the Om(2D) gene encodes a novel protein containing histidine/proline repeats, and is ubiquitously expressed during embryogenesis. The Om(2D) RNA is not detected in wild-type eye imaginal discs, but is abundantly found in the center of the eye discs of Om(2D) mutants, where excessive cell death occurs. D. melanogaster flies transformed with the Om(2D) cDNA under control of the hsp70 promoter display abnormal eye morphology when heat-shocked at the third larval instar stage. These results suggest that the Om(2D) gene is not normally expressed in the eye imaginal discs, but its ectopic expression, induced by the tom element, in the eye disc of third instar larvae results in defects in adult eye morphology.

Amino Acid Sequence↗
PubMed2004

Mutations affecting retinotectal axonal pathfinding in Medaka, Oryzias latipes.

We screened for mutations affecting retinotectal axonal projection in Medaka, Oryzias latipes. In wild-type Medaka embryos, all the axons of retinal ganglion cells (RGCs) project to the contralateral tectum, such that the topological relationship of the retinal field is maintained. We labeled RGC axons using DiI/DiO at the nasodorsal and temporoventral positions of the retina, and screened for mutations affecting the pattern of stereotypic projections to the tectum. By screening 184 mutagenized haploid genomes, seven mutations in five genes causing defects in axonal pathfinding were identified, whereas mutations affecting the topographic projection of RGC axons were not found. The mutants were grouped into two classes according to their phenotypes. In mutants of Class I, a subpopulation of the RGC axons branched out either immediately after leaving the eye or after reaching the midline, and this axonal subpopulation projected to the ipsilateral tectum. In mutants of Class II, subpopulations of RGC axons branched out after crossing the midline and projected aberrantly. These mutants will provide clues to understanding the functions of genes essential for axonal pathfinding, which may be conserved or partly divergent among vertebrates.

Animals↗
PubMed1998

Cancer prevention: past, present, future.

There are many examples of different types of cancers that have been prevented by appropriate measures in the past. Most of them were related to occupational, iatrogenic or accidental factors, often as the outcome of heavy exposure of humans to specific carcinogenic agents. Cancer is a disease of DNA, and is generally associated with multiple genetic alterations, these being produced in the typical case by exposure to various carcinogens, each of which exists at minute concentrations. Thus, the impact of carcinogenic factors, xenobiotics and autobiotics, is due to their actions in concert. However, a single mutation yielding genomic instability exerts a disproportionately large influence by resulting in a large number of secondary mutational events. Epigenetic changes can also not be disregarded especially from the view point of prevention of neoplasia. The occurrence of multiple primary cancers among survivors of initial primaries, and the presence of hereditary groups with a high risk of cancer development provide a strong stimulus for establishment of effective approach for cancer prevention, which should be, in principle, multi-faceted. Therefore, a holistic approach is essential with improvement in life style including choosing a balanced diet and avoidance of cigarette smoking and other sources of carcinogens, as integral elements.

Anticarcinogenic Agents↗
PubMed1998

EMS-induced polygenic mutation rates for nine quantitative characters in Drosophila melanogaster.

Polygenic mutations were induced by treating Drosophila melanogaster adult males with 2.5 mM EMS. The treated second chromosomes, along with untreated controls, were then made homozygous, and five life history, two behavioral, and two morphological traits were measured. EMS mutagenesis led to reduced performance for life history traits. Changes in means and increments in genetic variance were relatively much higher for life history than for morphological traits, implying large differences in mutational target size. Maximum likelihood was used to estimate mutation rates and parameters of distributions of mutation effects, but parameters were strongly confounded with one another. Several traits showed evidence of leptokurtic distributions of effects and mean effects smaller than a few percent of trait means. Distributions of effects for all traits were strongly asymmetrical, and most mutations were deleterious. Correlations between life history mutation effects were positive. Mutation parameters for one generation of spontaneous mutation were predicted by scaling parameter estimates from the EMS experiment, extrapolated to the whole genome. Predicted mutational coefficients of variation were in good agreement with published estimates. Predicted changes in means were up to 0.14% or 0.6% for life history traits, depending on the model of scaling assumed.

Analysis of Variance↗
PubMed1982

Resistance of herpes simplex virus to adenine arabinoside and E-5-(2-bromovinyl)-2'-deoxyuridine: a physical analysis.

The physical map limits of DNA sequences within the DNA polymerase locus of herpes simplex virus (HSV) type 1 that define resistance mutations to adenine arabinoside (ara-A) and E-5-(2-bromovinyl)-2'-deoxyuridine (BVDU) were determined. Intertypic recombinants between HSV types 1 and 2, generated by marker rescue of temperature-sensitive mutations, had genome structures determined by restriction endonuclease analysis and were used to show that the resistance mutation for ara-Ar-1 is closely linked to the resistance mutations for phosphonoacetic acid (paar-1) and acycloguanosine (acvr-1) within a region of 2.6 kilobase pairs (kbp) in the HSV DNA polymerase locus. The resistance mutation for bvdur-1 is defined by a 2.9-kbp region that overlaps with the region defining resistance to the other three drugs but that is transferred separately. The DNA polymerase locus also contains a 2.2-kbp region that maps adjacent to and to the left of the region defining the bvdur-1 mutation which can be transferred separately and defines a region determining the HSV-1-specific sensitivity to BVDU in a manner analogous to that to acyclovir.

Acyclovir↗
PubMed1995

Mutational analysis of the herpes simplex virus virion host shutoff protein: evidence that vhs functions in the absence of other viral proteins.

Herpes simplex virus (HSV) virions contain one or more factors that trigger rapid shutoff of host protein synthesis and accelerated decay of cellular and viral mRNAs in infected cells. HSV isolates bearing mutations at the virion host shutoff (vhs) locus (gene UL41) are defective for both processes, indicating that the vhs protein is required; however, it is not clear whether the role of vhs in shutoff is direct or indirect and if other virion components are also necessary. We therefore used a transient-cotransfection assay to determine if the vhs protein displays activity in the absence of other viral gene products. We found that a vhs expression vector strongly suppressed expression of a cotransfected lacZ reporter gene and that this effect was eliminated by the vhs1 point mutation that abolishes virion-induced host shutoff during HSV infection. Further evidence for the biological relevance of the transfection assay came from the demonstration that five vhs in-frame linker insertion mutations yielded concordant results when assayed in cotransfected cells and following transfer into the viral genome: three mutations eliminated activity in both assays, while two had no effect. On the basis of these results, we conclude that the vhs protein can trigger host shutoff in the absence of other HSV proteins. The cotransfection assay was used to rapidly assess the activities of a panel of linker insertion mutants spanning the vhs polypeptide. All mutations that mapped to regions conserved among the vhs homologs of alphaherpesvirus inactivated function; in contrast, four of five mutations that mapped to regions that are absent from several vhs homologs had no effect. These results further support the biological relevance of the transfection assay and begin to delineate functional domains of the vhs polypeptide.

Amino Acid Sequence↗
PubMed2006

Simple sequence proteins in prokaryotic proteomes.

BACKGROUND: The structural and functional features associated with Simple Sequence Proteins (SSPs) are non-globularity, disease states, signaling and post-translational modification. SSPs are also an important source of genetic and possibly phenotypic variation. Analysis of 249 prokaryotic proteomes offers a new opportunity to examine the genomic properties of SSPs. RESULTS: SSPs are a minority but they grow with proteome size. This relationship is exhibited across species varying in genomic GC, mutational bias, life style, and pathogenicity. Their proportion in each proteome is strongly influenced by genomic base compositional bias. In most species simple duplications is favoured, but in a few cases such as Mycobacteria, large families of duplications occur. Amino acid preference in SSPs exhibits a trend towards low cost of biosynthesis. In SSPs and in non-SSPs, Alanine, Glycine, Leucine, and Valine are abundant in species widely varying in genomic GC whereas Isoleucine and Lysine are rich only in organisms with low genomic GC. Arginine is abundant in SSPs of two species and in the non-SSPs of Xanthomonas oryzae. Asparagine is abundant only in SSPs of low GC species. Aspartic acid is abundant only in the non-SSPs of Halobacterium sp NRC1. The abundance of Serine in SSPs of 62 species extends over a broader range compared to that of non-SSPs. Threonine(T) is abundant only in SSPs of a couple of species. SSPs exhibit preferential association with Cell surface, Cell membrane and Transport functions and a negative association with Metabolism. Mesophiles and Thermophiles display similar ranges in the content of SSPs. CONCLUSION: Although SSPs are a minority, the genomic forces of base compositional bias and duplications influence their growth and pattern in each species. The preferences and abundance of amino acids are governed by low biosynthetic cost, evolutionary age and base composition of codons. Abundance of charged amino acids Arginine and Aspartic acid is severely restricted. SSPs preferentially associate with cell surface and interface functions as opposed to metabolism, wherein proteins of high sequence complexity with globular structures are preferred. Mesophiles and Thermophiles are similar with respect to the content of SSPs. Our analysis serves to expand the commonly held views on SSPs.

Amino Acid Sequence↗
PubMed2005

Neighboring-nucleotide effects on the mutation patterns of the rice genome.

DNA composition dynamics across genomes of diverse taxonomy is a major subject of genome analyses. DNA composition changes are characteristics of both replication and repair machineries. We investigated 3,611,007 single nucleotide polymorphisms (SNPs) generated by comparing two sequenced rice genomes from distant inbred lines (subspecies), including those from 242,811 introns and 45,462 protein-coding sequences (CDSs). Neighboring-nucleotide effects (NNEs) of these SNPs are diverse, depending on structural content-based classifications (genome-wide, intronic, and CDS) and sequence context-based categories (A/C, A/G, A/T, C/G, C/T, and G/T substitutions) of the analyzed SNPs. Strong and evident NNEs and nucleotide proportion biases surrounding the analyzed SNPs were observed in 1-3 bp sequences on both sides of an SNP. Strong biases were observed around neighboring nucleotides of protein-coding SNPs, which exhibit a periodicity of three in nucleotide content, constrained by a combined effect of codon-related rules and DNA repair mechanisms. Unlike a previous finding in the human genome, we found negative correlation between GC contents of chromosomes and the magnitude of corresponding bias of nucleotide C at -1 site and G at +1 site. These results will further our understanding of the mutation mechanism in rice as well as its evolutionary implications.

Base Composition↗