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Ten families of variant genes encoded in subtelomeric regions of multiple chromosomes of Plasmodium chabaudi, a malaria species that undergoes antigenic variation in the laboratory mouse.

The chromosome ends of human malaria parasites harbour many genes encoding proteins that are exported to the surface of infected red cells, often being involved in host-parasite interactions and immune evasion. Unlike other murine malaria parasites Plasmodium chabaudi undergoes antigenic variation during passage in the laboratory mouse and hence is a model suitable for investigation of switching mechanisms. However, little is known about the subtelomeric regions of P. chabaudi chromosomes and its variable antigens. Here we report 80 kb of sequence from an end of one P. chabaudi chromosome. Hybridization of probes spanning this region to two dimensional pulsed field gels of the genome revealed 10 multicopy gene families located exclusively in subtelomeric regions of multiple P. chabaudi chromosomes, interspersed amongst multicopy intergenic regions. Hence all chromosomes share a common subtelomeric structure, presumably playing a similar role in spatial positioning as the P. falciparum Rep20 sequence. Expression in blood stages, domains characteristic of surface antigens and copy numbers between four and several hundred per genome, indicate a functional role in antigenic variation for some of these families. We identify members of the cir family, as well as novel genes, that although clearly homologous to cir have large low complexity regions in the predicted extracellular domains. Although all families have homologues in other rodent Plasmodium species, four were previously not known to be subtelomeric. Six have homologues in human and simian malarias.

Animals↗

Screening of genetic and expression alterations of SRC1 gene in prostate cancer.

BACKGROUND: Genetic alterations of the SRC1 gene have not been thoroughly studied in prostate cancer. MATERIALS AND METHODS: Five prostate cancer cell lines and 32 xenografts were screened for mutations and gene copy number alterations. Subsequently, frequencies of detected sequence variations were further analyzed in 44 clinical prostate cancers, 6 benign prostate hyperplasias, and 48 normal controls. Finally, the protein expression of SRC1 in 254 clinical prostate tumors was investigated. RESULTS: Three non-recurrent sequence variations, and one single nucleotide polymorphism in the coding region of SRC1, as well as one case of SRC1 gene amplification were found. The protein expression of SRC1 was higher in androgen ablation resistant than untreated prostate carcinomas, but the difference was not statistically significant (P = 0.0796). CONCLUSIONS: Genetic alterations of SRC1 are rare in prostate cancer. The nuclear protein accumulation of SRC1 seems to be mildly increased in androgen ablation resistant prostate cancers. .

Androgen Antagonists↗

Different antifolate-resistant L1210 cell variants with either increased or decreased folylpolyglutamate synthetase gene expression at the level of mRNA transcription.

L1210 cell variants selected in the presence of the lipophilic dihydrofolate reductase inhibitor, metoprine, expressed increased levels of one-carbon, reduced folate transport inward (Sirotnak, F. M., Moccio, D. M., and Yang, C.-H. (1984) J. Biol. Chem. 259, 13139-13144). Growth of one of these variants (L1210/R69), with metoprine in the presence of decreasing concentrations of 1,L5-CHO-folateH4 (natural diastereoisomer of 5-formyl-tetrahydrofolate), resulted in the selection of other variants (L1210/R82, R83, and R84) with further reduction in one-carbon, reduced folate transport and in two cases (L1210/R83 and R84) with 3-8-fold increased folylpolyglutamate synthetase (FPGS) activity and folate compound polyglutamate formation in situ. Metoprine resistance was further increased, and the requirement for exogenous folate during growth was decreased as well in these variants. The increase in FPGS activity observed in L1210/R83 and R84 was characterized by 3- and 8-fold increases in value for Vmax with no change in Km and the same increase in a 60-61-kDa protein as shown by immunoblotting. Northern blotting revealed the same increases in these two variants in the level of a 2.3-kilobase FPGS mRNA when compared with control, while Southern blotting of genomic DNA did not reveal any increase in FPGS gene-copy number or restriction polymorphisms. Also, no difference in stability of FPGS mRNA was found between parental and variant cells. In contrast, nuclear run-on assays revealed differences among these cell types in the rate of FPGS mRNA transcription that correlated with increased FPGS activity, protein, and mRNA level in the variants. Similar studies with a transport-defective, methotrexate-resistant L1210 cell variant (L1210/R25) documented a 2-3-fold decrease in FPGS activity, protein, and mRNA levels that was accounted for by a decrease in FPGS mRNA transcription. These results provide the first examples of constitutively altered transcriptional regulation of FPGS activity associated with acquired resistance to antifolates.

Amino Acid Sequence↗

Study on the evolution of the grande retrotransposon in the zea genus.

The study of Grande retrotransposon (RTN) variation reported here comprises the intrinsic element variability and the changes that element insertion provokes in the Zea genome, including its abundance among species. Sequence analysis of a defined long-terminal repeat (LTR) region from Grande RTN revealed a high level of sequence divergence since no identical sequences were found among the 65 clones examined that belong to different Zea species or maize inbred lines. Average diversity values within accessions ranged from 0.17 to 0.37 substitutions per nucleotide. Phylogenetic analysis revealed a lack of concordance between the phylogenetic tree obtained from LTR sequences and the conventional taxonomic tree, suggesting that different subfamilies of Grande elements existed before Zea speciation. When sequence-specific amplification polymorphism (SSAP) marker data, which combines genomic and RTN variation, are used, the derived trees reflect the established species phylogeny and allow, as well, differentiating among some maize lines. Finally, the evaluation of Grande abundance, using different element probes in all the Zea species but Z. luxurians, revealed around 5,700 copies per haploid genome in all the diploid species examined, indicating a similar expansion process of Grande in all the Zea genomes. This number of copies represents in all cases around a 3% of the genome, which implies that Grande RTN is an important component of the maize genome. The copy number ratio LTR/gag is around 2 in all the species analyzed, indicating that overwhelming majority of elements have internal region. Thus, mechanisms such as homologous recombination between LTRs of a single RTN, which would remove the internal region and one LTR, leaving behind a single recombinant LTR, seems not to be active in maize for Grande RTN.

Base Sequence↗

Variation of microsatellite size homoplasy across electromorphs, loci, and populations in three invertebrate species.

Size homoplasy was analyzed at microsatellite loci by sequencing electromorphs, that is, variants of the same size (base pairs). This study was conducted using five interrupted and/or compound loci in three invertebrate species, the honey bee Apis mellifera, the bumble bee Bombus terrestris, and the freshwater snail Bulinus truncatus. The 15 electromorphs sequenced turned out to hide 31 alleles (i.e., variants identical in sequence). Variation in the amount of size homoplasy was detected among electromorphs and loci. From one to seven alleles were detected per electromorph, and one locus did not show any size homoplasy in both bee species. The amount of size homoplasy was related to the sequencing effort, since the number of alleles was correlated with the number of copies of electromorphs sequenced, but also with the molecular structure of the core sequence at each locus. Size homoplasy within populations was detected only three times, meaning that size homoplasy was detected mostly among populations. We analyzed population structure, estimating Fst and a genetic distance, based on either electromorphs or alleles. Whereas little difference was found in A. mellifera, uncovering size homoplasy led to a more marked population structure in B. terrestris and B. truncatus. We also showed in A. mellifera that the detection of size homoplasy may alter phylogenetic reconstructions.

Alleles↗

Bovine herpesvirus 1 isolates contain variable copy numbers of GC-rich tandem repeats in the gI non-coding regions of their genomes.

A polymerase chain reaction (PCR) targeted to the central portion of the bovine herpesvirus 1 (BHV1) genome, and overlapping the 3' untranslated end of the gI glycoprotein, was used to amplify BHV1 genomic sequences. PCR products generated from cell cultures infected with BHV1.1 were consistently smaller than the corresponding products from cells infected with BHV1.2. The nature of the sequence differences between these isolates within the target region was found to be a consequence of variable numbers of small GC rich repeats, particularly the sequence 5'-G(A/T)CC-3', present in the region downstream of the gI coding region. Based on these differences a modified PCR protocol which readily discriminated between several BHV1.1 and BHV1.2 strains was devised.

Animals↗

Copy number of P elements, KP/full-sized P element ratio and their relationships with environmental factors in Brazilian Drosophila melanogaster populations.

The P transposable element copy numbers and the KP/full-sized P element ratios were determined in eight Brazilian strains of Drosophila melanogaster. Strains from tropical regions showed lower overall P element copy numbers than did strains from temperate regions. Variable numbers of full-sized and defective elements were detected, but the full-sized P and KP elements were the predominant classes of elements in all strains. The full-sized P and KP element ratios were calculated and compared with latitude. The northernmost and southernmost Brazilian strains showed fewer full-sized elements than KP elements per genome, and the strains from less extreme latitudes had many more full-sized P than KP elements. However, no clinal variation was observed. Strains from different localities, previously classified as having P cytotype, displayed a higher or a lower proportion of KP elements than of full-sized P elements, as well as an equal number of the two element types, showing that the same phenotype may be produced by different underlying genomic components of the P-M system.

Animals↗

Factors contributing to the hybrid dysgenesis syndrome in Drosophila virilis.

A hybrid dysgenesis syndrome in Drosophila virilis is associated with the mobilization of at least four unrelated transposable elements designated Helena, Paris, Penelope and Ulysses. We carried out 42 crosses between eight strains differing in transposable element copy number in order to assess their contributions to hybrid dysgenesis. Linear regression and stepwise regression analysis was performed to estimate the correlation between the difference in euchromatic transposable element number between the parental flies of different strains involved in the crosses and the percentage, in the progeny of these crosses, of males with atrophic gonads. Male gonadal atrophy is a typical manifestation of the D. virilis hybrid dysgenesis syndrome. About half the variability in the level of male gonadal atrophy can be attributed to Penelope and Paris/Helena. Other factors also seem to play a significant role in hybrid dysgenesis in D. virilis, including maternally transmitted host factors and/or uncontrolled environmental variation. In the course of this work a novel transposable element named Telemac was found. Telemac is also mobilized in hybrid dysgenesis but does not appear to play a major causative role.

Animals↗

Gyrase activity and number of copies of the gyrase B subunit gene in Haemophilus influenzae.

Gyrase activities in extracts of various strains of Haemophilus influenzae can differ by more than an order of magnitude (J. K. Setlow, E. Cabrera-Juárez, W. L. Albritton, D. Spikes, and A. Mutschler, J. Bacteriol. 164:525-534, 1985). Measurements of in vitro activity and copy number indicated that most of these differences arose from variations in the number of copies of the gene for the gyrase B subunit, with some strains containing multicopy plasmids coding for that subunit. The quantitative relationship between gyrase and copy number depended on the mutations in the plasmids and in the host. The gyrase and copy number were considerably lower in plasmid-bearing strains carrying the prophage HP1c1. Two mutations affecting gyrase that are apparently regulatory caused an increase in gyrase without a concomitant increase in copy number. The possibility that the in vivo gyrase activity did not reflect the in vitro data was explored by measurement of alkaline phosphatase and ATPase activity in the extracts. Alkaline phosphatase activity increased with increasing gyrase activity measured in vitro, but ATPase activity did not. We conclude that extra supercoiling enhanced transcription of the alkaline phosphatase gene but not the ATPase gene and that it is unlikely that there is much discrepancy between gyrase activity assayed in vitro and the activity in the cell.

Adenosine Triphosphatases↗

[Cytoplasmic male sterility-associated structural variation of the mitochondrial genome regions containing rps3 and orf215 in sugar beet Beta vulgaris L].

Several 5'-degenerate primers were selected by computer analysis and used for mtDNA typing in sugar beet cultivars with cytoplasms of the S (typical for cytoplasmic male sterility) or N (normal) type. A number of N- or S-specific markers were found to correspond to transcribed mitochondrial genes. One was from the orf215 region of the N-type mtDNA. A physical map of the corresponding region was constructed for the S-type mtDNA, and a substantial difference observed for the two genome types. One N-specific marker proved to contain a rearranged rps3 region and a truncated atp9 copy. With the known nucleotide sequence of this marker, three-primer PCR was designed and showed that both variants of the rps3 region simultaneously take place in the mtDNA pool, the new one occurring in a substochiometric proportion.

Base Sequence↗

Replication stress increases de novo CNVs across the malaria parasite genome.

Changes in the copy number of large genomic regions, termed copy number variations (CNVs), contribute to important phenotypes. CNVs are readily identified using conventional approaches when present in a large fraction of the cell population. However, CNVs in only a few genomes are often overlooked but important; if beneficial, a de novo CNV that arises in a single genome can expand during selection to create a population of cells with novel characteristics. While single cell methods for studying de novo CNVs are increasing, we continue to lack information about CNV dynamics in rapidly evolving microbial populations. Here, we investigated de novo CNVs in the genome of the Plasmodium parasite that causes human malaria. The highly AT-rich P. falciparum genome readily accumulates CNVs that facilitate rapid adaptation. We employed low-input genomics and specialized computational tools to evaluate the impact of sub-lethal stress on the de novo CNV rate. We observed a significant increase in genome-wide de novo CNVs following treatment with an antimalarial compound that inhibits replication. De novo CNVs encompassed genes from various cellular pathways participating in human infection. This snapshot of CNV dynamics emphasizes the connection between replication stress, DNA repair, and CNV generation in this important microbial pathogen.

Journal Article↗

A novel arrangement of sequence elements surrounding the rDNA promoter and its spacer duplications in tsetse species.

Variation in organization and sequence of the rDNA of six species of tsetse fly (Glossina) has been investigated. Several novel tsetse-specific features have been uncovered. Like many other species the spacer is composed of subrepeats, which in some species contain duplications of the true promoter at the spacer-ETS boundary. In tsetse, however, the first 90 base-pairs of the external transcribed spacer (ETS) (that is, +1 to +90 after transcription initiation) is the 3' end of the last subrepeat. The absence of a "unique" region between the last subrepeat and the ETS suggests that the tsetse rDNA unit may consist of multiple true promoters, that is there is no single ETS boundary. Furthermore, interspecific comparisons show that the 90 base-pair region is part of a conserved 202 base-pair region, consisting of 72 base-pairs upstream from the initiation site and a further 40 base-pairs downstream, which is shared by all promoters other than the last. In genera other than tsetse, subrepeat lengths between species are generally similar; in tsetse they differ due to (1) variation in copy-number of the subsubrepeat motif A9T6CAG, and (2) the presence of large regions flanked by direct simple repeats such as GA5 or TGGTCTC. Slippage-like mechanisms are probably responsible for (1), and recombination and subsequent excision involving the direct repeats for (2). Different structural and sequence variants are seen to be homogenized in the family and fixed in each species, reflecting continual unequal crossing-over. However, notwithstanding this process of differentiation, the available comparisons also reveal that there are two small conserved regions between Glossina and Drosophila: one is part of the promoter and the other is an ETS processing site. Such intergeneric and interspecific differences are discussed in relation to the problem of the maintenance of several essential functions within the rDNA repeating unit despite the continual differentiation of the unit into novel arrangements.

Animals↗

Expression of Kirsten murine sarcoma virus in transformed nonproducer and revertant NIH/3T3 cells: evidence for cell-mediated resistance to a viral oncogene in phenotypic reversion.

Expression of the provirus in a clonally related series of Kirsten murine sarcoma virus-transformed NIH/3T3 nonproducer cell lines was examined at both the transcriptional and translational levels. All cells expressed high levels of genome-sized viral RNA with little variation between cell lines despite differences in provirus integration site and copy number. Expression of K-ras RNA was estimated to be at least 10- to 20-fold higher than that of the mouse cellular homolog of the viral transforming gene. Levels of the virus-coded transforming protein, p21, were similarly elevated, with little variation between nonproducer cells. In two revertant cell lines containing a normal provirus and a rescuable transforming gene, no impairment in expression at either the transcriptional or translational level was found. After superinfection with Kirsten murine sarcoma virus, one revertant became more tumorigenic, whereas the other remained nontumorigenic. These results show that cell transformation by Kirsten murine sarcoma virus is invariably associated with elevated expression of the virus-coded oncogene and that one of the revertants is resistant to the action of the viral transforming gene.

Animals↗

Low and homogeneous copy number of plasmid-borne symbiont genes affecting host nutrition in Buchnera aphidicola of the aphid Uroleucon ambrosiae.

The bacterial endosymbiont of aphids, Buchnera aphidicola, often provides amino acids to its hosts. Plasmid amplification of leucine (leuABCD) and tryptophan (trpEG) biosynthesis genes may be a mechanism by which some Buchnera over-produce these nutrients. We used quantitative polymerase chain reaction to assess the leuABCD/trpEG copy variability within Uroleucon ambrosiae, an aphid with a wide diet breadth and range. Both leuABCD and trpEG abundances are: (i) similar for aphids across 15 populations, and (ii) low compared to Buchnera from other aphid species (particularly trpEG). Consequently, the plasmid location of trpEG combined with Buchnera's chromosomal polyploidy may functionally limit, rather than increase, tryptophan production within Uroleucon ambrosiae.

Animals↗

Copy number lability and evolutionary dynamics of the Adh gene family in diploid and tetraploid cotton (Gossypium).

Nuclear-encoded genes exist in families of various sizes. To further our understanding of the evolutionary dynamics of nuclear gene families we present a characterization of the structure and evolution of the alcohol dehydrogenase (Adh) gene family in diploid and tetraploid members of the cotton genus (Gossypium, Malvaceae). A PCR-based approach was employed to isolate and sequence multiple Adh gene family members, and Southern hybridization analyses were used to document variation in gene copy number. Adh gene copy number varies among Gossypium species, with diploids containing at least seven Adh loci in two primary gene lineages. Allotetraploid Gossypium species are inferred to contain at least 14 loci. Intron lengths vary markedly between loci, and one locus has lost two introns usually found in other plant Adh genes. Multiple examples of apparent gene duplication events were observed and at least one case of pseudogenization and one case of gene elimination were also found. Thus, Adh gene family structure is dynamic within this single plant genus. Evolutionary rate estimates differ between loci and in some cases between organismal lineages at the same locus. We suggest that dynamic fluctuation in copy number will prove common for nuclear genes, and we discuss the implications of this perspective for inferences of orthology and functional evolution.

Alcohol Dehydrogenase↗

Matrix attachment regions (MARs) enhance transformation frequencies and reduce variance of transgene expression in barley.

Nuclear matrix attachment regions (MARs) are defined as genomic DNA sequences, located at the physical boundaries of chromatin loops. They are suggested to play a role in the cis unfolding and folding of the chromatin fibre associated with the regulation of gene transcription. Inclusion of MARs in transgene cassettes enhances their expression and reduces position-effect variations in the transgenic host. The present study is the first to investigate the influence of MAR sequences on transformation frequencies and transgene expression in barley, which is highly relevant to the future improvement of this crop by biotechnology. Two plant MAR sequences were tested both for their ability to bind to the nuclear matrix of barley leaf nuclei and to regulate the expression of a reporter gene in transgenic barley. Competitive in vitro MAR binding assays with the 520 bp P1-MAR from soybean and the 516 bp TBS-MAR from petunia revealed that only the P1-MAR had specific binding affinity for barley nuclear matrices. The barley transformation frequency with the uidA reporter gene was increased 2-fold when the gene was flanked with either the P1-MAR or TBS-MAR, while the gene copy number was strongly reduced. The presence of P1-MAR sequences increased the mean activity and reduced the variance in expression of a co-integrated reporter gene in barley consistent with the proposed model of MAR activity.

Cells, Cultured↗

Mitochondrial D-loop sequences are integrated in the rat nuclear genome.

We have cloned two fragments of rat nuclear DNA (nucDNA), 3.3 x 10(3) nucleotide-pairs (knp) and 9.1 knp, that contain a 0.5 knp section sharing 80% sequence identity with the mitochondrial DNA (mtDNA) heavy strand origin of replication (D-loop) nascent strand and 88% identity with each other. The light and heavy strand promoters of the D-loop region are not present in either clone, thus they likely do not function as replication origins in the nuclear genome. The nucDNA sequences surrounding the mtDNA-like sequences are not mitochondrial, thus the mtDNA-like sequences are demonstrably covalently linked in the nuclear genome. Indeed, the surrounding nuclear sequences of each clone also share 88% identity. This sequence arrangement strongly suggests an initial insertion of mtDNA into nucDNA with subsequent amplification of an encompassing region of nucDNA. Divergence calculations suggest that the mtDNA insertion occurred around 13.6 million years ago (MYA) with the subsequent separation occurring around 6.5 MYA. The mtDNA-like sequences of the nuclear clones hybridize strongly to a number of different BamHI-PstI restriction fragments, suggesting either repeated integration and/or frequent mutational events producing new restriction enzyme sites. It is not yet known if one or more of the uncloned D-loop-like sequences are associated with promoters, which would suggest possible function. The 3.3 knp nucDNA fragment is present in low copy number. In contrast, the 9.1 knp nucDNA fragment appears to be moderately repeated. The elements do not appear to be tandemly repeated. The nucDNA clones contain remnants of rat long interspersed repetitive element (LINE) sequences; in addition the 9.1 knp fragment contains sequences with similarity to portions of viral reverse transcriptase and RNaseH genes. Until now, all mtDNA-like sequences found in the nuclear genome have been coding sequences. This is the first confirmation by sequence analysis of a portion of the mtDNA control region in the nuclear genome.

Amino Acid Sequence↗

Evaluation of Two Methods for Quantitation of Hepatitis C Virus RNA.

Background: Accurate quantitation of hepatitis C virus (HCV) RNA in serum may provide a means to predict disease course and response to interferon-alpha therapy. Several quantitative assays are commercially available, but none have been accepted as the gold standard. Methods and Results: The branched DNA quantitative hybridization assay (Quantiplex HCV 1.0, Chiron, Emeryville, CA) and a quantitative reverse transcription polymerase chain reaction (Amplicor HCV MONITOR, Roche Diagnostic Systems, Branchburg, NJ) were compared using a panel of 53 sera from patients with chronic hepatitis C. All sera contained HCV RNA of known genotype. Overall, there was a positive correlation between the results for the 41 sera that gave discrete values in both tests (r =.81, linear regression; P <.01, Kendall's rank test); however, the mean number of HCV copies per milliliter was 13.5-fold higher with Quantiplex (P <.01). A plot of the difference between methods against their means showed poor agreement between the methods. No correlation between the results of the two tests was observed for sera with MONITOR values greater than 5.0 x 10(5) copies/mL. Discrete MONITOR values were obtained for all 12 sera that were below the lower limit of quantitation of Quantiplex (mean, 1.78 x 10(5)). Parallel testing of serial dilutions of two sera showed that each method gave linear responses over the stated dynamic ranges; however, the proportional systematic error was greater with MONITOR. The mean coefficient of variation for replicate determinations was 23% for Quantiplex and 45% for MONITOR (P =.13). Conclusions: Despite a positive correlation, systematic differences exist between the two methods for quantitation of HCV RNA and they cannot be used interchangeably.

Journal Article↗