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PubMed1982

Isolation and characterization of human DNA fragments with nucleotide sequence homologies with the simian virus 40 regulatory region.

A recombinant library of human DNA sequences was screened with a segment of simian virus 40 (SV40) DNA that spans the viral origin of replication. One hundred and fifty phage were isolated that hybridized to this probe. Restriction enzyme and hybridization analyses indicated that these sequences were partially homologous to one another. Direct DNA sequencing of two such SV40-hybridizing segments indicated that this was not a highly conserved family of sequences, but rather a set of DNA fragments that contained repetitive regions of high guanine plus cytosine content. These sequences were not members of the previously described Alu family of repeats and hybridized to SV40 DNA more strongly than do Alu family members. Computer analyses showed that the human DNA segments contained multiple homologies with sequences throughout the SV40 origin region, although sequences on the late side of the viral origin contained the strongest cross-hybridizing sequences. Because of the number and complexity of the matches detected, we could not determine unambiguously which of the many possible heteroduplexes between these DNAs was thermodynamically most favored. No hybridization of these human DNA sequences to any other segment of the SV40 genome was detected. In contrast, the human DNA segments isolated cross-hybridized with many sequences within the human genome. We tested for the presence of several functional domains on two of these human DNA fragments. One SV40-hybridizing fragment, SVCR29, contained a sequence which enhanced the efficiency of thymidine kinase transformation in human cells by approximately 20-fold. This effect was seen in an orientation-independent manner when the sequence was present at the 3' end of the chicken thymidine kinase gene. We propose that this segment of DNA contains a sequence analogous to the 72-base-pair repeats of SV40. The existence of such an "activator" element in cellular DNA raises the possibility that families of these sequences may exist in the mammalian genome.

Base Composition↗

PubMed1997

The vibrator mutation causes neurodegeneration via reduced expression of PITP alpha: positional complementation cloning and extragenic suppression.

The mouse vibrator mutation causes an early-onset progressive action tremor, degeneration of brain stem and spinal cord neurons, and juvenile death. We cloned the vibrator mutation using an in vivo positional complementation approach and complete resequencing of the resulting 76 kb critical region from vibrator and its parental chromosome. The mutation is an intracisternal A particle retroposon insertion in intron 4 of the phosphatidylinositol transfer protein alpha gene, causing a 5-fold reduction in RNA and protein levels. Expression of neurofilament light chain is also reduced in vibrator, suggesting one signaling pathway that may underlie vibrator pathology. The vibrator phenotype is suppressed in one intercross. We performed a complete genome scan and mapped a major suppressor locus (Mvb-1) to proximal chromosome 19.

Alleles↗

PubMed2001

A genome screen for multiple sclerosis in Italian families.

We have screened the whole genome for linkage in 40 Italian multiplex families with multiple sclerosis using 322 markers. The GENEHUNTER-PLUS program was used to analyse these data and revealed eight regions of potential linkage where the lod score exceeds the nominal 5% significance level (0.7). No region of linkage with genome-wide significance was identified and none of the markers showed evidence of statistically significant transmission disequilibrium. Overall these results have refined the linkage data relating to this disease in Italians modestly supporting some previously identified areas of interest and helping to exclude others.

Genetic Testing↗

PubMed1993

Cis-preferential replication of the turnip yellow mosaic virus RNA genome.

The largest open reading frame of the turnip yellow mosaic virus RNA genome encodes a 206-kDa protein that is cleaved to yield N-terminal 150-kDa (p150) and C-terminal 70-kDa (p70) proteins. Using a genomic cDNA clone capable of generating infectious transcripts in vitro, we have introduced substitution, frameshift, and in-frame deletion mutations into the regions encoding both proteins. None of the mutant RNAs was able to replicate independently in turnip protoplasts, indicating that p150 and p70 are both essential. The replication in protoplasts of most of these defective RNAs was poorly supported in trans by a coinoculated helper genome with a deletion in the coat protein gene; replication could also be supported in trans by certain defective RNAs, but this complementation was likewise inefficient in most cases. The replication in trans was more efficient for defective RNAs encoding wild-type p150 and defective p70 than for those encoding defective p150 and wild-type p70. One defective RNA with a large deletion in the p70 coding region was able to replicate efficiently, both when inoculated with the helper genome and when inoculated with a second complementing defective RNA that supplied a wild-type p70. Thus, the cis preference of replication can be overcome in some cases. A model in which p150 and p70 form a complex with the 3' end of the RNA is proposed to explain the cis-preferential replication of turnip yellow mosaic virus RNA.

Amino Acid Sequence↗

PubMedDate not supplied

An indexed genomic library for Paramecium complementation cloning.

Recent pioneering work opened the way for cloning genes in Paramecium by functional complementation of mutants. We present here the construction and pilot utilization of a new indexed library of Paramecium macronuclear DNA. The library is made of 61,440 clones containing inserts mostly between 6 and 12 kilobases. It has already allowed the complementation cloning of four new genes, and this library has proven to be very useful for rapid hybridization cloning.

Animals↗

PubMed2001

Genomic analysis of some Japanese isolates of Getah virus.

Using the reverse transcription-polymerase chain reaction (RT-PCR) and direct sequencing, capsid protein and non-structural protein 1 (nsP1) regions of Sagiyama virus and eight Getah virus strains were analysed. The viruses were isolated from Malaysia and various areas of Japan over a period of 30 years. Based on the available published sequence data, oligonucleotide primers were designed for RT-PCR and the sequences were determined. Our findings showed that though there were differences in the nucleotide sequences in the nsP1 region, there was 100% amino acid homology. On the other hand, in the capsid region, the nucleotide differences caused a major difference in the amino acid sequence. Therefore, the difference in the capsid region is one of the useful markers in the genetic classification between Sagiyama virus and strains of Getah virus, and might be responsible for the serological difference in complement fixation test. The genomic differences among the Getah virus strains are due to time factor rather than geographical distribution.

Alphavirus↗

PubMed1994

Characterisation of the S-adenosylmethionine decarboxylase (SAMDC) gene of potato.

S-adenosylmethionine decarboxylase (SAMDC) is involved in the biosynthesis of the polyamines, spermidine and spermine. Recently, we reported the isolation of a putative cDNA clone of the SAMDC clone of potato (Plant Mol Biol 20; 641-651). In order to confirm that the potato genes does encode SAMDC, a complementation experiment with a yeast strain that possesses a null mutation in the SAMDC gene was performed. The yeast strain contains a deletion-insertion mutation in the SAMDC gene and has an absolute requirement for the addition of exogenous spermidine for growth. When the full-length potato cDNA was expressed in the mutant yeast strain there was no longer a requirement for exogenous spermidine. Immunoblotting experiments suggest that the potato SAMDC gene product has an apparent molecular mass of 39 kDa. Expression of the SAMDC gene was high in the young and actively dividing tissues and low in the mature and non-dividing tissues of both vegetative and reproductive organs. Additionally, isolation and characterisation of the corresponding genomic clone is reported. The gene has one intron in its 5'-untranslated sequence but otherwise the transcribed portion is identical to the cDNA clone.

Adenosylmethionine Decarboxylase↗

PubMed2002

A genome screen for linkage disequilibrium in HLA-DRB1*15-positive Germans with multiple sclerosis based on 4666 microsatellite markers.

Multiple sclerosis (MS) is a demyelinating disorder of the central nervous system with putative autoimmune aetiology and complex genetic background. Here, we report the results of a genome screen for linkage disequilibrium (LD) by using 6000 microsatellite markers in 198 HLA-DRB1*15-positive MS patients and 198 unrelated controls (pooled DNA); 4666 analysed markers could be included in the resulting association map, from which 87 revealed significant differences between MS cases and controls.

Adult↗

PubMed2002

Molecular analysis of de novo pyrimidine synthesis in solanaceous species.

The de novo synthesis of pyrimidine nucleotides in plants has been analysed on a molecular level with special focus on cDNA cloning and structure analysis of all genes involved and their expression pattern during development. The exhaustive cloning of all cDNAs resulted from screening with heterologous cDNAs or by using complementation strategies with Escherichia coli mutants and subsequent enzyme activity measurements. Southern hybridization and comparison with the Arabidopsis genome reveals plant specific aspects and a simple genomic organization of pyrimidine synthesis in plants, which is superimposed by the postulated, complex subcellular compartmentalization. Northern hybridization evinces coordinated expression of all genes under developmental control during tobacco leaf growth.

Arabidopsis↗

PubMed1998

Construction and characterization of a 10-fold genome equivalent rat P1-derived artificial chromosome library.

A rat PAC library was constructed in the vector pPAC4 from genomic DNA isolated from female Brown Norway rats. This library consists of 215,409 clones arrayed in 614,384-well microtiter plates. An average insert size of 143 kb was estimated from 217 randomly isolated clones, thus representing approximately 10-fold genome coverage. This coverage provides a very high probability that the library contains a unique sequence in genome screening. Tests on randomly selected clones demonstrated that they are very stable, with only 4 of 130 clones showing restriction digest fragment alterations after 80 generations of serial growth. FISH analysis using 70 randomly chosen PACs revealed no significant chimeric clones. About 7% of the clones analyzed contained repetitive sequences related to centromeric regions that hybridized to some but not all centromeres. DNA plate pools and superpools were made, and high-density filters each containing an array of 8 plates in duplicate were prepared. Library screening on these superpools and appropriate filters with 10 single-locus rat markers revealed an average of 8 positive clones, in agreement with the estimated high genomic coverage of this library and representation of the rat genome. This library provides a new resource for rat genome analysis, in particular the identification of genes involved in models of multifactorial disease. The library and high-density filters are currently available to the scientific community.

Animals↗

PubMed2003

Environmental biotechnology in the postgenomics era.

The sequencing of the human genome and many microbial genomes has provided new opportunities to study the environmental impact on life processes, leading to development of new technologies that can be protected by patenting. Development of such new technologies has, however, led in some cases to judicial intervention because of their controversial nature. This article illustrates some of the trends in postgenomics biotechnology development and the attendant legal and ethical considerations.

Biotechnology↗

PubMed2004

Community structure and metabolism through reconstruction of microbial genomes from the environment.

Microbial communities are vital in the functioning of all ecosystems; however, most microorganisms are uncultivated, and their roles in natural systems are unclear. Here, using random shotgun sequencing of DNA from a natural acidophilic biofilm, we report reconstruction of near-complete genomes of Leptospirillum group II and Ferroplasma type II, and partial recovery of three other genomes. This was possible because the biofilm was dominated by a small number of species populations and the frequency of genomic rearrangements and gene insertions or deletions was relatively low. Because each sequence read came from a different individual, we could determine that single-nucleotide polymorphisms are the predominant form of heterogeneity at the strain level. The Leptospirillum group II genome had remarkably few nucleotide polymorphisms, despite the existence of low-abundance variants. The Ferroplasma type II genome seems to be a composite from three ancestral strains that have undergone homologous recombination to form a large population of mosaic genomes. Analysis of the gene complement for each organism revealed the pathways for carbon and nitrogen fixation and energy generation, and provided insights into survival strategies in an extreme environment.

Archaea↗

PubMed2005

Large-scale genomic approaches to brain development and circuitry.

Over the past two decades, molecular genetic studies have enabled a common conceptual framework for the development and basic function of the nervous system. These studies, and the pioneering efforts of mouse geneticists and neuroscientists to identify and clone genes for spontaneous mouse mutants, have provided a paradigm for understanding complex processes of the vertebrate brain. Gene cloning for human brain malformations and degenerative disorders identified other important central nervous system (CNS) genes. However, because many debilitating human disorders are genetically complex, phenotypic screens are difficult to design. This difficulty has led to large-scale, genomic approaches to discover genes that are uniquely expressed in brain circuits and regions that control complex behaviors. In this review, we summarize current phenotype- and genotype-driven approaches to discover novel CNS-expressed genes, as well as current approaches to carry out large-scale, gene-expression screens in the CNS.

Animals↗

PubMedDate not supplied

[The identification of alphaviruses by using cDNA probes complementary to the 3'-terminal of the viral genome].

Venezuelan equine encephalomyelitis, Eastern equine encephalomyelitis and Sindbis viruses, as well as members of various serological groups of alphaviruses were identified using synthesized molecular probes complementary to 3'-terminus of the molecule of the appropriate genomic RNA: 32P-single-stranded kDNA and E. coli-cloned 32P-double-stranded kDNA. A high species-specificity of kDNA-probes was demonstrated. The sensitivity of the method was 1 ng of viral RNA overlayed on the nitrocellulose filter.

Alphavirus↗

PubMed2003

Genome-wide mRNA profiling reveals heterochronic allelic variation and a new imprinted gene in hybrid maize endosperm.

We have taken a genomic approach to examine global gene expression in the maize endosperm in relation to dosage and parental effects. Endosperm of eight hybrids generated by reciprocal crosses and their seven inbred parents were sampled at three developmental stages: 10, 14, and 21 days after pollination (DAP). These samples were subjected to GeneCalling, an open-ended mRNA-profiling technology, which simultaneously analyzes thousands of genes. Results indicated that the overall level of gene expression in the maize endosperm was dosage-dependent, that is, the gene expression was proportional to the parental genome contribution of 2n maternal : 1n paternal. However, approximately 8% of the genes deviated from such allelic additive expression and exhibited differential expression in hybrids of reciprocal crosses, resembling either maternally or paternally expressed genes. There were more genes with maternal-like expression (MLE) than those with paternal-like expression (PLE). Allele-specific expression analysis of four selected genes using the WAVE denaturing HPLC (dHPLC) system revealed several mechanisms responsible for the deviation from the allelic additive expression in the hybrid endosperm: heterochronic allelic variation, allelic variation in the level of expression, and genomic imprinting. We discovered a novel imprinted gene no-apical-meristem (NAM) related protein1 (nrp1) that was expressed only in the endosperm and regulated by gene-specific imprinting. The nrp1 gene, a putative transcriptional factor, may play an important role in endosperm development.

Alleles↗

PubMed1995

Molecular evolution of the hepatitis B virus genome.

The hepatitis B virus (HBV) has a circular DNA genome of about 3,200 base pairs. Economical use of the genome with overlapping reading frames may have led to severe constraints on nucleotide substitutions along the genome and to highly variable rates of substitution among nucleotide sites. Nucleotide sequences from 13 complete HBV genomes were compared to examine such variability of substitution rates among sites and to examine the phylogenetic relationships among the HBV variants. The maximum likelihood method was employed to fit models of DNA sequence evolution that can account for the complexity of the pattern of nucleotide substitution. Comparison of the models suggests that the rates of substitution are different in different genes and codon positions; for example, the third codon position changes at a rate over ten times higher than the second position. Furthermore, substantial variation of substitution rates was detected even after the effects of genes and codon positions were corrected; that is, rates are different at different sites of the same gene or at the same codon position. Such rates after the correction were also found to be positively correlated at adjacent sites, which indicated the existence of conserved and variable domains in the proteins encoded by the viral genome. A multiparameter model validates the earlier finding that the variation in nucleotide conservation is not random around the HBV genome. The test for the existence of a molecular clock suggests that substitution rates are more or less constant among lineages. The phylogenetic relationships among the viral variants were examined. Although the data do not seem to contain sufficient information to resolve the details of the phylogeny, it appears quite certain that the serotypes of the viral variants do not reflect their genetic relatedness.

Codon↗

PubMed2002

Ordered cosmid library of the Mesorhizobium loti MAFF303099 genome for systematic gene disruption and complementation analysis.

For effective exploitation of the genome sequence information of Lotus microsymbiont, Mesorhizobium loti MAFF303099, to discover gene functions, we have constructed an ordered and mutually overlapping cosmid library using an IncP broad host-range vector. The library consisted of 480 clones to cover approximately 99.6% of the genome with average insert size and overlap of 26.9 and 11.1 kbp, respectively. The genome of M. loti consists of a single chromosome and two plasmids. The chromosome (7,036,071 bp) was covered 99.68% by 445 clones with four gaps, although two clones were unstable in E. coli. The larger plasmid pMLa (351,911 bp) was completely covered by 23 clones, while the smaller pMLb (208,315 bp) was covered 98.85% by 12 clones with two gaps. We have also made ancillary plasmids to facilitate the construction of deletion mutants using derivatives of the library clones. As a pilot experiment to uncover regions which contain novel symbiotic genes, 13 deletion mutants were constructed to lack in total 180.5 kbp of the genome. All the mutants formed apparently normal nodules and supported symbiotic nitrogen fixation, however, one mutant that lacked a 5.3 kbp chromosomal region, 4,551,930-4,557,222, did not produce normal exopolysaccharides as judged by fluorescence on medium containing Calcofluor. The results supported the effectiveness of the approach to detect gene functions.

Cosmids↗

PubMed1999

Systematic search for susceptibility genes in different populations.

We analyzed the first replicate of each of the four simulated population samples from three distinct populations by linkage and association genome scans and could identify three regions with susceptibility loci for the disease: on chromosome 1, marker D1G024, with strong evidence for gene x environment interaction; or chromosome 3, around marker D3G045; and on chromosome 5, markers D5G035-D5G042. Our results were obtained without knowing the true disease model and are compared with this model in the discussion.

Alleles↗