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PubMed2006

Independent occurrences of multiple repeats in the control region of mitochondrial DNA of white-tailed deer.

Deer in the genera Mazama and Odocoileus generally have two copies of a 75-base-pair (bp) repeat in the left domain of the control region of the mitochondrial DNA (mtDNA). Phylogenetic analyses further suggest an ancient origin for the duplication supporting a previously stated contention that this event occurred before the separation of Mazama and Odocoileus. However, white-tailed deer (Odocoileus virginianus) had three or four copies of a 75-bp repeat in the control region of their mtDNA in 7.8% of the individuals analyzed, and all of these animals were from the coastal plain of the southeastern United States. When copy 3 is present, it is very similar in sequence to copy 2, but variation suggests that copy 3 probably evolved multiple times from copy 2. The pattern of phylogenetic clustering of the haplotypes from across the coastal plain also suggests that phenotypes with three or four copies of the repeat have originated multiple times. The 44 observed haplotypes showed strong spatial subdivision across the area with subpopulations frequently showing complete shifts in haplotype frequencies from others taken from nearby areas. Many of the subpopulations right along the coast or on adjacent barrier islands have a limited number of haplotypes as would occur in populations undergoing drift because of small numbers of breeding females and limited female dispersal.

Animals↗

PubMed2002

Polymorphism in the subtelomeric regions of chromosomes of Kinetoplastida.

Leishmania spp. and the related kinetoplastid Trypanosoma brucei are single-celled parasites. In Leishmania, the nuclear genome comprises 36 diploid chromosomes and occasional amplified minichromosomes, while the T. brucei nucleus contains 11 larger diploid chromosomes and a variable number of intermediate-sized and minichromosomes. This paper primarily describes the subtelomeric structure of the larger diploid chromosomes of L. major and T. brucei, although some aspects may also apply to smaller chromosomes. The diploid chromosomes contain most protein-coding genes and vary in size. The telomeric sequence is common to both species, but adjacent subtelomeric repeats vary between species and chromosomes. It is possible that some of the complex repeats described here play a role in stabilizing replication and copy number of the chromosomes. The subtelomeric regions of T. brucei chromosomes differ from those of other protozoan parasites, as they are dedicated to expression sites for variant surface glycoprotein genes, used by the parasite to evade immune destruction by antigenic variation. Variation in these sites creates segmental aneuploidy in many T. brucei chromosomes.

Animals↗

PubMedDate not supplied

Chromosomal localization of DNA amplifications in neuroblastoma tumors using cDNA microarray comparative genomic hybridization.

Conventional comparative genomic hybridization (CGH) profiling of neuroblastomas has identified many genomic aberrations, although the limited resolution has precluded a precise localization of sequences of interest within amplicons. To map high copy number genomic gains in clinically matched stage IV neuroblastomas, CGH analysis using a 19,200-feature cDNA microarray was used. A dedicated (freely available) algorithm was developed for rapid in silico determination of chromosomal localizations of microarray cDNA targets, and for generation of an ideogram-type profile of copy number changes. Using these methodologies, novel gene amplifications undetectable by chromosome CGH were identified, and larger MYCN amplicon sizes (in one tumor up to 6 Mb) than those previously reported in neuroblastoma were identified. The genes HPCAL1, LPIN1/KIAA0188, NAG, and NSE1/LOC151354 were found to be coamplified with MYCN. To determine whether stage IV primary tumors could be further subclassified based on their genomic copy number profiles, hierarchical clustering was performed. Cluster analysis of microarray CGH data identified three groups: 1) no amplifications evident, 2) a small MYCN amplicon as the only detectable imbalance, and 3) a large MYCN amplicon with additional gene amplifications. Application of CGH to cDNA microarray targets will help to determine both the variation of amplicon size and help better define amplification-dependent and independent pathways of progression in neuroblastoma.

Algorithms↗

PubMed2006

Copy number of bean mitochondrial genes estimated by real-time PCR does not correlate with the number of gene loci and transcript levels.

Structural rearrangements characteristic for plant mitochondrial DNA often result in the appearance of genes in new genomic environments. The determination of the real number of gene copies is difficult since the in vivo structure of plant mitochondrial genomes is questionable. It is still uncertain whether the gene copy number regulates transcription in plant mitochondria. Using the real-time PCR technique we have quantified the copies of mitochondrial genes and their transcripts in four related Phaseolus vulgaris lines. We found low intergenomic variation both in the copy number of particular genes and the abundance of their transcripts, while the intragenomic differences between copy numbers and transcripts levels of various genes were much higher. Furthermore, we found that the appearance of a gene in a new location is not correlated with a proportional increase in its copy number estimated by real-time PCR. This observation seems to result from gene dosage compensation which is probably associated with the multimolecular plant mitochondrial genome structure and particularly with the recombinogenic activity of large repeats. Based on the relative gene copy numbers we propose the existence of two types of Phaseolus mitochondrial genomes: one associated with fertility and the other inducing cytoplasmic male sterility. We also show that there is no correlation between the observed number of copies of the analyzed genes and the steady-state level of their transcripts.

Fertility↗

PubMed1994

Nucleotide sequence variation of chitin synthase genes among ectomycorrhizal fungi and its potential use in taxonomy.

DNA sequences of single-copy genes coding for chitin synthases (UDP-N-acetyl-D-glucosamine:chitin 4-beta-N-acetylglucosaminyltransferase; EC 2.4.1.16) were used to characterize ectomycorrhizal fungi. Degenerate primers deduced from short, completely conserved amino acid stretches flanking a region of about 200 amino acids of zymogenic chitin synthases allowed the amplification of DNA fragments of several members of this gene family. Different DNA band patterns were obtained from basidiomycetes because of variation in the number and length of amplified fragments. Cloning and sequencing of the most prominent DNA fragments revealed that these differences were due to various introns at conserved positions. The presence of introns in basidiomycetous fungi therefore has a potential use in identification of genera by analyzing PCR-generated DNA fragment patterns. Analyses of the nucleotide sequences of cloned fragments revealed variations in nucleotide sequences from 4 to 45%. By comparison of the deduced amino acid sequences, the majority of the DNA fragments were identified as members of genes for chitin synthase class II. The deduced amino acid sequences from species of the same genus differed only in one amino acid residue, whereas identity between the amino acid sequences of ascomycetous and basidiomycetous fungi within the same taxonomic class was found to be approximately 43 to 66%. Phylogenetic analysis of the amino acid sequence of class II chitin synthase-encoding gene fragments by using parsimony confirmed the current taxonomic groupings. In addition, our data revealed a fourth class of putative zymogenic chitin synthesis.

Amino Acid Sequence↗

PubMed1989

Integration of multiple copies of a foreign sequence into the Ti plasmid of Agrobacterium tumefaciens.

A method for constructing Ti plasmids bearing multiple copies of a sequence integrated in tandem is described. A small plasmid that confers tetracycline resistance (TcR), contains homology to a Ti plasmid, and is unable to replicate in Agrobacterium tumefaciens, was mobilized from Escherichia coli to A. tumefaciens. Ti plasmids of exconjugants selected for resistance to 12-14 micrograms Tc/ml all contained multiple tandem repeats of the integrative plasmid. Tc-sensitive variants with fewer integrated copies arose spontaneously at low frequency in the absence of Tc selection, or could be enriched for by selection on Tc in combination with the bactericidal antibiotic augmentin. Variants having an increased number of integrated copies were obtained by growth on high Tc concentrations. Tandem repeats integrated between border sequences provide, in principle, a way to reproducibly introduce many linked copies of any foreign gene into plants.

Blotting, Southern↗

PubMed1995

Altered expression after expansion of a v-erbA transgene in transgenic mice.

Repetitive DNA is known to undergo size variations based on an increase or decrease in the number of monomer units. We describe here the spontaneous expansion of an experimentally introduced tandem array of repeats in a transgenic mouse consisting of the human beta-actin promoter fused to the viral oncogene v-erbA and an SV40 polyadenylation signal (hAP/v-erbA). The expansion of the transgene was identified during routine screening of transgenic offspring for heterozygosity or homozygosity in one founder line. The control heterozygote genome consisted of the hAP/v-erbA transgene organized as a tandem array approximately five monomer units at a single chromosomal locus. The number of units increased to 20-21 copies, and germline transmission of the expanded units was stable for at least two generations. The majority of animals carrying the expanded monomer units retained their RNA expression patterns; however, some of these animals had drastically reduced expression levels. We discuss the possibility that expansion of repeated units may provide a mechanism by which the expression of a deleterious transgene is reduced.

Actins↗

PubMed1990

Leishmania major: expression and gene structure of the glycoprotein 63 molecule in virulent and avirulent clones and strains.

Two Leishmania membrane glycoconjugates, gp63 and lipophosphoglycan, have been implicated in parasite attachment and uptake into the host macrophage. Moreover, recent data suggest that parasite virulence is associated with high expression of gp63. In this study we have surveyed gp63 gene copy number, in addition to the level of expression of gp63 mRNA and protein in several Leishmania major isolates, as well as virulent and avirulent strains and clones. The highest level of gp63 expression was found in the avirulent cloned line LRC-L119.3G7, which expresses about a 15-fold higher level of gp63 RNA and protein than the virulent cloned line LRC-L137/7/V121, suggesting that large amounts of gp63 are not sufficient for infectivity and do not correlate with virulence. L119.3G7 has eight copies of the gp63 gene compared to five copies in the virulent cloned line V121 and its parental virulent isolate LRC-L137. A series of avirulent clones derived from LRC-L137 also had five copies of the gene, suggesting that gp63 copy number is maintained among closely related parasites. Different virulent isolates of L. major from different geographic regions exhibited six copies of the gp63 gene. The variation in total gene copy number is due to different numbers of the tandemly repeated gp63 isogene in different strains. Our data show that there is wide variability between strains of L. major in the copy number of gp63 genes as well as in the amount of RNA and protein expressed.

Animals↗

PubMed2000

The 5S rRNA gene diversity in Kengyilia rigidula (Keng and S.L. Chen) J.L. Yang, Yen, and Baum (Poaceae: Triticeae): possible contribution of the H genome to the origin of Kengyilia.

Fifty-three units of 5S rDNA sequences from five accessions of Kengyilia rigidula, a member of the tribe Triticeae that also includes wheat, barley, rye, and their wild relatives, have been amplified by the polymerase chain reaction (PCR), cloned, and sequenced. The genome of K. rigidula consists of three haplomes, St, P, and Y. An evaluation of the aligned sequences of the diverse 53 different 5S DNA units yielded three 5S-unit classes. One unit class, Long S1, was assignable to the St haplome, one unit class, the Long P1, was assignable to the P haplome, and a third unit class, Long H1, was assignable to the H haplome. The last was expected to be assignable to the Y haplome, based on previous knowledge. Evolutionary scenarios are put forward to explain this finding. Among those possibilities is that the number of copies of units assignable to the Y haplome is very small and difficult to detect. Short units, reported earlier in K. alatavica, were not found in K. rigidula.

Base Sequence↗

PubMed2003

The non-clonal and transitory nature of HIV in vivo.

From a posteriori analyses of genetic variation, recombination can only be identified when the parental genomes are distinct. For viruses like HIV-1, this requires the producer cell to be infected by more than one virus. Using fluorescence in situ hybridisation, the provirus copy numbers in splenocytes from two HIV-1 patients were determined. More than 75% of infected splenocytes harboured two or more proviruses, range 1-8, with a mean of approximately 3-4 per cell. Sequencing of amplified DNA from single laser micro-dissected cells showed an extraordinary degree of diversity while numerous recombinants were evident. Given the dynamics of HIV-1 turnover in vivo and a recombination rate of approximately 3 cross-overs per cycle, some genomes from a fifteen year old infection may have undergone as many cross-overs as bases in the genome. Thus, recombination profoundly influences HIV evolution and gives it a non-clonal and transitory nature in vivo.

HIV Infections↗

PubMed1997

Genetic variation in the dimorphic regions of RAP-1 genes and rap-1 loci of Babesia bigemina.

The rhoptry-associated protein-1 (RAP-1) of Babesia bigemina induces protective immune responses in cattle. RAP-1 has two regions of sequence dimorphism at the carboxy and amino terminal ends, respectively. Neutralization-sensitive, surface-exposed B-cell epitopes are present in the amino terminal variant type 1 (NT-1), and CD4+ T-cell epitopes in the carboxy terminal variant type 1 (CT-1). Importantly, antibodies recognizing NT-1 epitopes do not cross react with NT-2 and CD4+ T-cells recognizing epitopes in CT-1 do not cross react with CT-2, suggesting that variation in dimorphic regions of RAP-1 is immunologically significant. We evaluated rap-1 locus structure and the extent of sequence variation in the dimorphic regions of rap-1 genes from geographically diverse strains of B. bigemina. All strains contained NT-1 and NT-2 the encoding sequences were highly conserved, with at least 99%, nucleotide identity among strains. However, the Puerto Rico strain encoded a hybrid NT-1/NT-2 sequence which appears to have originated by a gene conversion event. The 3' ends of rap-1 genes, which include the carboxy terminal variants, are conserved among strains. A new and conserved CT variant (CT-3), with a region of sequence identity to CT-2 and a sequence not related to either CT-1 or CT-2, was identified in all strains of B. bigemina. All but one strain encode both NTs and the three CT variants. The S1A strain, an attenuated strain from Argentina, does not encode CT-2. While NT-1 is associated only with CT-1, NT-2 can be associated with all three CT variants in RAP-1. Within the genome, rap-1 genes are arranged in tandem repeats but with different gene copy number and arrangements among strains. Collectively, the data suggest that gene conversion and unequal recombination events contribute to overall rap-1 sequence conservation among gene variants and strains but may also generate new rap-1 variants.

Amino Acid Sequence↗

PubMed1998

The tomato Cf-5 disease resistance gene and six homologs show pronounced allelic variation in leucine-rich repeat copy number.

The tomato Cf-2 and Cf-5 genes confer resistance to Cladosporium fulvum and map to a complex locus on chromosome 6. The Cf-5 gene has been isolated and is predicted to encode a largely extracytoplasmic protein containing 32 leucine-rich repeats (LRRs), resembling the previously isolated Cf-2 gene, which has 38 LRRs. Three haplotypes of this locus from Lycopersicon esculentum, L. pimpinellifolium, and L. esculentum var cerasiforme were compared, and five additional homologs of Cf-5 were sequenced. All share extensive sequence identity, particularly within the C-terminal portions of the predicted proteins. In striking contrast to the Cf-9 gene family, six of seven homologs in the Cf-2/Cf-5 gene family vary in LRR copy number, ranging from 25 to 38 LRRs. Cf-5 and one adjacent homolog differ by only two LRRs. Recombination events that vary the LRR copy number in this region could provide a mechanism for the generation of new specificities for recognition of different ligands. A recombination breakpoint between the Cf-2 and Cf-5 loci was fully characterized and shown to be intragenic.

Alleles↗

PubMed1995

Analysis of pfmdr1 and drug susceptibility in fresh isolates of Plasmodium falciparum from subsaharan Africa.

Resistance of Plasmodium falciparum to many therapeutic agents is an increasing problem in most endemic areas. The role of the mdr-like gene products of P. falciparum in resistance to quinoline-containing compounds is not clear. The purpose of this study was to further examine the role of pfmdr1 in drug resistance in fresh clinical isolates originating from Africa. Drug susceptibility testing (chloroquine, mefloquine, halofantrine and quinine) and a molecular analysis of pfmdr1 was completed for 51 fresh clinical isolates. A statistical association between the chloroquine sensitivity phenotype and an intragenic allele of pfmdr1 was noted at a position, amino acid 86, which was previously associated with chloroquine resistance. There was little variation in the other intragenic alleles previously associated with chloroquine resistance. No correlation between pfmdr1 intragenic allelic variation and susceptibility to mefloquine, halofantrine or quinine was found. There was no association between gene copy number of pfmdr1 and any drug resistant phenotype in an analysis of selected isolates. This, along with other data, suggests that mefloquine resistance may have arisen by two different mechanisms in African and Southeast Asian isolates. Much more variability in the polyasparaginated region of the pfmdr1 gene was noted in this study than previously reported. In addition, fingerprint analysis using multiplex PCR revealed considerable genetic variability among these isolates.

Africa South of the Sahara↗

PubMed1994

Flow-cytometric quantification in human gliomas of alpha satellite DNA sequences specific for chromosome 7 using fluorescence in situ hybridization.

Cell suspensions prepared from freshly frozen tissue specimens were used to examine aberrations in the number of chromosome 7 signals in 10 human gliomas. Nuclear DNA was hybridized in vitro with an alpha satellite DNA probe specific for the centromeric regions of chromosome 7, using fluorescence in situ hybridization (FISH). The intensity of the fluorescence signal from the hybridized probe was measured, together with the nuclear DNA content, by flow cytometry. The mean probe fluorescence of all nuclei was compared to the mean copy number per nucleus found with microscopic scoring. Moreover, the mean probe fluorescence ratio of DNA aneuploid nuclei relative to DNA diploid nuclei (FISHa/FISHd) was calculated to determine how the numerical aberration of chromosome 7 signals contributes to the DNA ploidy of the sample. The results from the flow-cytometric analysis and from microscopic evaluation were compatible. One of four tumors with DNA diploidy had a higher average intensity of FISH signal and a broader coefficient of variation in FISH signal than normal brain tissue; this was shown to be due to the gain of chromosome 7 signals. Although FISHa/FISHd correlated with DNA indices (P < 0.01), there were some disparities, probably due to other complex genotypic associations involving several gains or losses of chromosomes. Thus gain of chromosome 7 in gliomas is related to both DNA ploidy change and chromosome specific gain. It is concluded that flow-cytometric quantification of FISH is useful in investigating numerical aberrations of chromosomes and nuclear DNA content simultaneously.

Aneuploidy↗

PubMed1993

A family of IS1031 elements in the genome of Acetobacter xylinum: nucleotide sequences and strain distribution.

An insertion sequence (here called IS1031A) from Acetobacter xylinum ATCC 23769 has recently been isolated. This study describes the complete nucleotide sequence of IS1031A as well as the sequences of two novel iso-IS1031 elements, IS1031C and IS1031D, from A. xylinum ATCC 23769. The three ISs are all exactly 930 bp long, have imperfect terminal inverted repeats of 24 bp for IS1031A and 21 bp for IS1031C and IS1031D, are flanked by three base pair direct repeats, and contain an open reading frame encoding a putative basic protein of 278 amino acids. Because of nucleotide substitutions, IS1031C and IS1031D differ from IS1031A by 12.9% while IS1031C differs from IS1031D by only 0.6%. Hybridization analyses of total DNA from nine A. xylinum strains showed that all strains contained IS1031-like elements varying in copy number from three to at least 16. None of three Acetobacter aceti strains examined contained IS1031-like elements. Taken together, the results suggest that A. xylinum contains a family of IS1031 elements with considerably diversified nucleotide sequences.

Amino Acid Sequence↗

PubMed1987

Distribution and abundance of insertion sequences among natural isolates of Escherichia coli.

A reference collection of 71 natural isolates of Escherichia coli (the ECOR collection) has been studied with respect to the distribution and abundance of transposable insertion sequences using DNA hybridization. The data include 1173 occurrences of six unrelated insertion sequences (IS1, IS2, IS3, IS4, IS5 and IS30). The number of insertion elements per strain, and the sizes of DNA restriction fragments containing them, is highly variable and can be used to discriminate even among closely related strains. The occurrence and abundance of pairs of unrelated insertion sequences are apparently statistically independent, but significant correlations result from stratifications in the reference collection. However, there is a highly significant positive association among the insertion sequences considered in the aggregate. Nine branching process models, which differ in assumptions regarding the regulation of transposition and the effect of copy number on fitness, have been evaluated with regard to their fit of the observed distributions. No single model fits all copy number distributions. The best models incorporate no regulation of transposition and a moderate to strong decrease in fitness with increasing copy number for IS1 and IS5, strong regulation of transposition and a negligible to weak decrease in fitness with increasing copy number for IS3, and less than strong regulation of transposition for IS2, IS4 and IS30.

Base Sequence↗

PubMed1994

Quantification of mRNA in human tissue using fluorescent nested reverse-transcriptase polymerase chain reaction.

We report the development of a quantitative nested reverse-transcriptase polymerase chain reaction which utilizes a fluorescence detection system. Using specific primer pairs to study mRNA for endothelin receptors in the human kidney, we synthesized a cRNA construct containing the same sequences but yielding a PCR product some 300 base pairs larger than native mRNA. Inclusion of a known amount of construct as internal standard with tissue RNA prior to cDNA synthesis allowed all reactions to occur under the same conditions in the same tube. In the nested PCR reaction, serial dilutions made before the second round enabled construction of a standard curve for each assay, and confirmation that standard and sample curves remained parallel. This indicates that both cDNAs amplified at the same rate. One internal primer was fluorescently labeled. Quantification of products using an ABI 373A sequencer with Genescan software gave sensitive and reproducible results. Analysis of a needle biopsy (10 mg) of histologically normal cortex gave 0.4 amol ETA mRNA and 1.6 amol ETB mRNA/micrograms total RNA. In medulla these values were 0.46 and 1.16 amol/micrograms, respectively. Ratios of ETB to ETA message were 74:26 in cortex and 77:23 in medulla, agreeing with previous ligand binding studies of receptor protein. Intra- and interassay coefficients of variation were 4.5 and 5.3%. This new method has potential for widespread application to the study of low copy-number mRNA or where only very small amounts of tissue are available, such as biopsy specimens.

Base Sequence↗

PubMed1985

Variation in myc gene amplification and expression in sublines of HL60 cells.

Three sublines of the human promyelocytic leukaemia cell line HL60 have been isolated. Histochemical, karyotypic and immunological analyses have confirmed the identity of the three sublines as HL60. However, the extent to which c-myc homologous DNA sequences are amplified in the genomes of these cells varies from 4- to 30-fold. Moreover, the relative abundance of c-myc-related RNA sequences in uninduced cells of each subline is directly proportional to the gene copy number. It is concluded that, while high levels of the c-myc gene and its transcripts may have had a role in the establishment of HL60 cells, these are not required for maintenance of the HL60 phenotype. However we cannot rule out the possibility that a low level of amplification and enhancement of transcription has a role in maintenance of this phenotype.

Antibodies, Monoclonal↗