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PubMed1980

Sequence complexity of cDNA transcribed from a diverse mRNA population.

Mouse liver poly(A)+mRNA was reverse transcribed using oligo-p(dT) or random oligonucleotides as primers to yield cDNA about equal to the mass of the template RNA. The size profile of the oligo-p(dT)-primedd cDNA was similar to that of the template RNA. RNA or cDNA driven saturation annealing of labeled single copy genomic DNA (scDNA) showed that 2% of the scDNA was complementary in either case indicating the sequence complexity of cDNA was equivalent to that of the template mRNA. These results establish for the first time that cDNA represents essentially all of the sequence complexity of a diverse template RNA population in which individual mRNA species are present in vastly different concentrations. RNA driven hydridization of the cDNA showed that about 40% of the cDNA mass represents most of the sequence complexity of the template RNA. Also, kinetics of this hybridization indicate a complexity of 58,000 kb for the template RNA, a value similar to that obtained by scDNA hybridization. We conclude that appropriately characterized cDNA probes can be used to make valid qualitative and quantitative comparisons of the complex, infrequent class mRNAs of different cells and tissues.

Animals↗

PubMed1991

Occurrence and stability of insertion sequences in Mycobacterium tuberculosis complex strains: evaluation of an insertion sequence-dependent DNA polymorphism as a tool in the epidemiology of tuberculosis.

In this study we established the usefulness of DNA fingerprinting for the epidemiology of tuberculosis on the basis of the DNA polymorphism generated by the insertion sequence (IS) IS986. Although clinical isolates of Mycobacterium tuberculosis displayed a remarkably high degree of restriction fragment length polymorphism, we showed that transposition of this IS element is an extremely rare event in M. tuberculosis complex strains grown either in vitro or in vivo for long periods of time. The M. tuberculosis and Mycobacterium africanum strains tested in this study contained 6 to 17 IS copies. In the Mycobacterium bovis strains, the copy numbers ranged between 1 and 5, and all 27 M. bovis BCG strains investigated invariably contained a single IS copy. This copy was located at a unique chromosomal position, reinforcing the idea that the frequency of IS transposition is very low in M. tuberculosis complex strains. Various microepidemics are described in which each microepidemic corresponds to a particular fingerprint type. The extent of similarity between Dutch and African strains was quantitatively assessed by computer-assisted analysis of DNA fingerprints. The results indicate that M. tuberculosis strains from regions in central Africa, where tuberculosis is highly prevalent, are generally more related to each other than isolates from the Netherlands, where the transmission rate is low and where the majority of the tuberculosis cases are presumed to be the result of reactivation of previously contracted M. tuberculosis infections.

Africa↗

PubMed1998

Isolation of an Arabidopsis thaliana cDNA by complementation of a yeast abc1 deletion mutant deficient in complex III respiratory activity.

The yeast Abc1 protein acts as a chaperone-like protein essential for the proper conformation and efficient functioning of the respiratory complex III. By functional complementation of a yeast abc1 mutant, we have identified an Arabidopsis thaliana cDNA that corresponds to a single copy gene and encodes a protein sharing 45% similarity with the yeast Abc1p protein. Cytochrome spectra and respiratory activity measurements have shown that the plant protein allows a partial restoration of the complex III activity. No major difference in the steady-state level of ABC1At mRNA was observed in various plant tissues, suggesting that ABC1At is constitutively expressed in A. thaliana. Phylogenetic analysis revealed that the Abc1At protein belongs to a large family of proteins composed of two eukaryotic and one prokaryotic subgroups differing by their degree of similarity and probably by their function.

Amino Acid Sequence↗

PubMed2003

Rho3p regulates cell separation by modulating exocyst function in Schizosaccharomyces pombe.

Cytokinesis is the final stage of the cell division cycle in which the mother cell is physically divided into two daughters. In recent years the fission yeast Schizosaccharomyces pombe has emerged as an attractive model organism for the study of cytokinesis, since it divides using an actomyosin ring whose constriction is coordinated with the centripetal deposition of new membranes and a division septum. The final step of cytokinesis in S. pombe requires the digestion of the primary septum to liberate two daughters. We have previously shown that the multiprotein exocyst complex is essential for this process. Here we report the isolation of rho3(+), encoding a Rho family GTPase, as a high-copy suppressor of an exocyst mutant, sec8-1. Overproduction of Rho3p also suppressed the temperature-sensitive growth phenotype observed in cells lacking Exo70p, another conserved component of the S. pombe exocyst complex. Cells deleted for rho3 arrest at higher growth temperatures with two or more nuclei and uncleaved division septa between pairs of nuclei. rho3Delta cells accumulate approximately 100-nm vesicle-like structures. These phenotypes are all similar to those observed in exocyst component mutants, consistent with a role for Rho3p in modulation of exocyst function. Taken together, our results suggest the possibility that S. pombe Rho3p regulates cell separation by modulation of exocyst function.

Actins↗

PubMed1990

The yeast genus Trichosporon spec. LS3;molecular characterization of genomic complexity.

The genomic structure of an industrial yeast strain Trichosporon spec. LS3 was compared with Trichosporon adeninovorans type strain CBS 8244. The cot values, the portion of single copy sequences (CBS 8244: 10.9 x 10(9) D, LS3: 10.6 x 10(9) D) as well as of repetitive sequences (CBS 8244: 6.0 x 10(9) D, LS3: 5.5 x 10(9) D) per haploid genome and genome complexity of these strains (CBS 2844: 16.9 x 10(9) D, LS3: 16.1 x 10(9) D) have been analysed. Both strains show a high genome complexity. The mitochondrial DNA content was measured and compared. No plasmidal DNA was identified for both strains. A survey of the data of genomic DNA made it possible to postulate for each of both strains a haploid set of chromosomes. The intraspecific reassoziation values of the nuclear DNA from the strain LS3 and CBS 8244 are interpreted to confirm classification of these strains based on physiological and genetical properties into one species Trichosporon adeninovorans.

Centrifugation, Density Gradient↗

PubMed2001

BrkA protein of Bordetella pertussis inhibits the classical pathway of complement after C1 deposition.

Bordetella pertussis produces a 73-kDa protein, BrkA (Bordetella resistance to killing), which inhibits the bactericidal activity of complement. In this study we characterized the step in the complement cascade where BrkA acts, using three strains: a wild-type strain, a strain containing an insertional disruption of brkA, and a strain containing two copies of the brkA locus. Following incubation with 10% human serum, killing was greatest for the BrkA mutant, followed by that for the wild-type strain, while the strain with two copies of brkA was the most resistant. Complement activation was monitored by enzyme-linked immunosorbent assay (ELISA) or Western blotting. ELISAs for SC5b-9, the soluble membrane attack complex, showed that production of SC5b-9 was greatest with the brkA mutant, less with the wild type, and least with the strain containing two copies of brkA. Deposition of complement proteins on the bacteria was monitored by Western blotting. A decrease in deposition on the bacteria of C4, C3, and C9 corresponded with decreased complement sensitivity. Deposition of C1, however, was not affected by the presence of BrkA. These studies show that BrkA inhibits the classical pathway of complement activation and prevents accumulation of deposited C4.

Bacterial Outer Membrane Proteins↗

PubMed2002

Transfer RNA gene-targeted retrotransposition of Dictyostelium TRE5-A into a chromosomal UMP synthase gene trap.

The genome of the eukaryotic microorganism Dictyostelium discoideum hosts a family of seven non-long terminal repeat retrotransposons (TREs) that show remarkable insertion preferences near tRNA genes. We developed an in vivo assay to detect tRNA gene-targeted retrotransposition of endogenous TREs in a reporter strain of D. discoideum. A tRNA gene positioned within an artificial intron was placed into the D. discoideum UMP synthase gene. This construct was inserted into the D. discoideum genome and presented as a landmark for de novo TRE insertions. We show that the tRNA gene-tagged UMP synthase gene was frequently disrupted by de novo insertions of endogenous TRE5-A copies, thus rendering the resulting mutants resistant to 5-fluoroorotic acid selection. Approximately 96% of all isolated 5-FOA-resistant clones contained TRE5-A insertions, whereas the remaining 4% resulted from transposition-independent mutations. The inserted TRE5-As showed complex structural variations and were found about 50 bp upstream of the reporter tRNA gene, similar to previously analysed genomic copies of TRE5-A. No integration by other members of the TRE family was observed. We found that only 51% of the de novo insertions were derived from autonomous TRE5-A.1 copies. The remaining 49% of new insertions were due to TRE5-A.2 elements, which lack the proteins required for reverse transcription and integration, but retain functional promoter sequences.

Animals↗

PubMedDate not supplied

DNA sequence at the end of IS1 required for transposition.

The insertion sequence IS1 belongs to a class of bacterial transposable genetic elements that can form compound transposons in which two copies of IS1 flank an otherwise non-transposable segment of DNA. IS1 differs from other known elements of this class (such as IS10, IS50 and IS903) in several respects. It is one of the smallest known insertion elements, exhibits a relatively complex array of open reading frames, is present in the chromosomes of various Enterobacteria, in some cases in many copies, and its insertion can result in the duplication of either 8 or 9 base pairs (bp) in the target DNA. Furthermore, although, like other members of the compound class, it seems to undergo direct transposition, IS1 also promotes replicon fusion (co-integrate formation) at a relatively high frequency. Like all other elements studied to date, the integrity of the extremities of IS1 are essential for efficient transposition. We have constructed a test system to determine the minimal DNA sequences at the extremities of IS1 required for transposition. Sequential deletions of the end sequences reveal that 21-25 bp of an isolated extremity are sufficient for transposition. A specific sequence 13-23 bp from the ends, defining the edge of the minimal sequence, is implicated as an essential site. The sites, symmetrically arrayed at both ends of IS1, correspond to the apparent consensus sequence of the known binding sites for the Escherichia coli DNA-binding protein (called integration host factor or IHF) which is required for the site-specific recombination that leads to integration of bacteriophage lambda into the bacterial genome. The sites at the ends of IS1 may thus bind a host protein, such as JHF or a related protein, that is involved in regulating the transposition apparatus.

Base Sequence↗

PubMed2003

The genetics and genomics of cancer.

The past decade has seen great strides in our understanding of the genetic basis of human disease. Arguably, the most profound impact has been in the area of cancer genetics, where the explosion of genomic sequence and molecular profiling data has illustrated the complexity of human malignancies. In a tumor cell, dozens of different genes may be aberrant in structure or copy number, and hundreds or thousands of genes may be differentially expressed. A number of familial cancer genes with high-penetrance mutations have been identified, but the contribution of low-penetrance genetic variants or polymorphisms to the risk of sporadic cancer development remains unclear. Studies of the complex somatic genetic events that take place in the emerging cancer cell may aid the search for the more elusive germline variants that confer increased susceptibility. Insights into the molecular pathogenesis of cancer have provided new strategies for treatment, but a deeper understanding of this disease will require new statistical and computational approaches for analysis of the genetic and signaling networks that orchestrate individual cancer susceptibility and tumor behavior.

Alleles↗

PubMed1988

Characterization of two operons encoding the cytochrome b6-f complex of the cyanobacterium Nostoc PCC 7906. Highly conserved sequences but different gene organization than in chloroplasts.

We have isolated and determined the nucleotide and derived protein sequences for the four genes, petCA and BD, which encode the cytochrome b6-f, electron-transfer complex of the filamentous cyanobacterium, Nostoc PCC 7906. The primary structure and cotranscription of the petCA genes encoding the Rieske-FeS (nuclear encoded in plants) and apocytochrome f proteins has been described previously (Kallas, T., Spiller, S., and Malkin, R. (1988) Proc. Natl. Acad. Sci. U.S.A., in press). The petBD genes (645 and 480 protein-coding nucleotides, respectively) for the apocytochrome b6 (24.3 kDa) and subunit-IV (17.5 kDa) proteins comprise a second operon located at least 12 kilobases (kb) from petCA. The Nostoc petBD genes are not closely linked to the psbB gene (encoding the 51-kDa photosystem II polypeptide) and do not contain introns as do the closely related chloroplast genes. DNA probes specific for each of the Nostoc cytochrome-complex genes hybridized to single bands in genomic DNA blots at intensities expected for single copy genes. These data suggest that a single set of cytochrome b6-f proteins function in the different types of membranes found in Nostoc vegetative and heterocyst cells. RNA blot hybridizations identified an 1.8-kb mRNA common to cytochrome b6 and subunit IV, and an intensely hybridizing 0.8-kb mRNA specific to the subunit IV gene probe. The role of the latter RNA is not clear but it may represent a transcript from the opposite strand. The deduced Rieske, apocytochrome f, apocytochrome b6, and subunit IV proteins exhibit 59, 58-63, 84-85, and 79-83% sequence identity with the proteins from chloroplast cytochrome b6-f complexes. The Nostoc proteins show lower but still significant sequences identity with the corresponding proteins of the mitochondrial-type b-c1 complexes. The four probable heme-liganding His residues, and the approximate spacings between them, have been conserved in all of the available cytochrome b6 and b sequences from divergent sources. The Nostoc apocytochrome b6 and subunit IV proteins, as well as the Rieske, appear to be translated and thus inserted into the membrane as mature forms without cleavable presequences. Hydropathy analyses revealed five potential membrane spans in cytochrome b6 and three in the subunit IV protein, consistent with the profiles observed for the chloroplast proteins and the related cytochrome b proteins of cytochrome b-c1 complexes.

Amino Acid Sequence↗

PubMed2003

Comparing the Rey and Taylor complex figures: a test-retest study in young and older adults.

The Rey and Taylor figures, two visual perception and memory tests, are not equivalent in terms of their difficulty. The purpose of this study was to examine factors such as age, gender, order of administration, copy time, and copy score that could account for the variability in recall performance. Copy score was shown to be the best overall predictor of recall. Younger participants recalled more units than older participants on both complex figures. Furthermore, older participants appeared to benefit more from the presentation of the Taylor figure first compared to when they were presented with the Rey figure first. The present findings reveal the importance of test order and provide equations that can be used to facilitate the comparison of scores derived from the two figures in test-retest situations.

Adult↗

PubMed2005

The vacuolar kinase Yck3 maintains organelle fragmentation by regulating the HOPS tethering complex.

The regulation of cellular membrane flux is poorly understood. Yeast respond to hypertonic stress by fragmentation of the normally large, low copy vacuole. We used this phenomenon as the basis for an in vivo screen to identify regulators of vacuole membrane dynamics. We report here that maintenance of the fragmented phenotype requires the vacuolar casein kinase I Yck3: when Yck3 is absent, salt-stressed vacuoles undergo fission, but reassemble in a SNARE-dependent manner, suggesting that vacuole fusion is disregulated. Accordingly, when Yck3 is deleted, in vitro vacuole fusion is increased, and Yck3 overexpression blocks fusion. Morphological and functional studies show that Yck3 modulates the Rab/homotypic fusion and vacuole protein sorting complex (HOPS)-dependent tethering stage of vacuole fusion. Intriguingly, Yck3 mediates phosphorylation of the HOPS subunit Vps41, a bi-functional protein involved in both budding and fusion during vacuole biogenesis. Because Yck3 also promotes efficient vacuole inheritance, we propose that tethering complex phosphorylation is a part of a general, switch-like mechanism for driving changes in organelle architecture.

Adaptor Proteins, Vesicular Transport↗

PubMed2005

Heterogeneous nuclear ribonucleoprotein C1/C2, MeCP1, and SWI/SNF form a chromatin remodeling complex at the beta-globin locus control region.

Locus control regions (LCRs) are regulatory DNA sequences that are situated many kilobases away from their cognate promoters. LCRs protect transgenes from position effect variegation and heterochromatinization and also promote copy-number dependence of the levels of transgene expression. In this work, we describe the biochemical purification of a previously undescribed LCR-associated remodeling complex (LARC) that consists of heterogeneous nuclear ribonucleoprotein C1/C2, nucleosome remodeling SWI/SNF, and nucleosome remodeling and deacetylating (NuRD)/MeCP1 as a single homogeneous complex. LARC binds to the hypersensitive 2 (HS2)-Maf recognition element (MARE) DNA in a sequence-specific manner and remodels nucleosomes. Heterogeneous nuclear ribonucleoprotein C1/C2, previously known as a general RNA binding protein, provides a sequence-specific DNA recognition element for LARC, and the LARC DNA-recognition sequence is essential for the enhancement of transcription by HS2. Independently of the initiation of transcription, LARC becomes associated with beta-like globin promoters.

Amino Acid Sequence↗

PubMed2001

Infectious RNA transcribed in vitro from a cDNA copy of the human coronavirus genome cloned in vaccinia virus.

The coronavirus genome is a positive-strand RNA of extraordinary size and complexity. It is composed of approximately 30000 nucleotides and it is the largest known autonomously replicating RNA. It is also remarkable in that more than two-thirds of the genome is devoted to encoding proteins involved in the replication and transcription of viral RNA. Here, a reverse-genetic system is described for the generation of recombinant coronaviruses. This system is based upon the in vitro transcription of infectious RNA from a cDNA copy of the human coronavirus 229E genome that has been cloned and propagated in vaccinia virus. This system is expected to provide new insights into the molecular biology and pathogenesis of coronaviruses and to serve as a paradigm for the genetic analysis of large RNA virus genomes. It also provides a starting point for the development of a new class of eukaryotic, multi-gene RNA vectors that are able to express several proteins simultaneously.

Animals↗

PubMedDate not supplied

Genomic gain of the epidermal growth factor receptor harboring band 7p12 is part of a complex pattern of genomic imbalances in oral squamous cell carcinomas.

BACKGROUND: To determine the association between changes of genomic gene dose and clinical parameters in squamous cell carcinomas of the oral cavity, comparative genomic hybridization seemed suited not only to detect genomic imbalances in these tumors, but also particularly to examine the role of gain of 7p12, the band harboring the epidermal growth factor receptor (EGFR) in this context. METHODS: Total genomic DNA obtained from 35 oral squamous cell carcinomas was subjected to comparative genomic hybridization (CGH) and detected patterns of genomic imbalances were associated with various clinical parameters. RESULTS: The examined tumors exhibited five and up to 47 DNA copy number alterations (CNAs). Nineteen of these showed a gain of chromosome band 7p12. A highly complex but strikingly consistent pattern of genomic imbalances (average, 32 CNAs per tumor) was associated with this alteration, among which gains clearly dominated over losses of genomic material. Comparable patterns, however, could also be found in a few tumors with a high number of CNAs (average, 26) but without the 7p gain. Low numbers of imbalances always were accompanied by low consistency of CNA patterns and none of these cases showed enh(7p12). No significant differences with respect to pT class or grade of tumors were found between enh(7p)-positive and -negative tumors. Stage IV and lymph node affection were slightly more frequent among enh(7p12)-positive than in -negative cases. Relapse occurred in 63% in 7p12-positive vs. 25% in the negative group. Average disease-free survival of tumors without 7p gain clearly exceeded that of tumors with gain of 7p (36.8 vs. 21.3). However, some of these associations could also be found if comparison was based on number of CNAs. By means of hierarchical cluster analysis, we were able to show that different patterns of CNAs can be separated from each other in tumors with or without 7p alterations, and that these patterns predict short- or long-term survival of patients. CONCLUSIONS: Previously described associations of gains of 7p12, the chromosomal band harboring the EGFR gene with clinical parameters can reasonably be estimated only within the context of the pattern and complexity of the genomic imbalances accompanying this chromosomal loss in examined tumors.

Adult↗

PubMed1998

Hydroxyl radical footprinting of DNA complexes of the ets domain of PU.1 and its comparison to the crystal structure.

Hydroxyl radical footprinting has been used to probe interactions in complexes between the ets domain of the murine transcription factor PU.1 and three different DNA restriction fragments, each containing one copy of the recognition sequence 5'-GGAA-3'. Two natural PU.1 binding sites, the SV40 enhancer site and the lambdaB motif of Ig lambda2-4 enhancer, were used as well as the PU.1 binding site present in the crystallized PU.1-DNA complex [Kodandapani, R., Pio, F., Ni, C.-Z., Piccialli, G., Klemsz, M., McKercher, S. R., Maki, R. A., and Ely, K. R. (1996) Nature 380, 456-460]. The footprints obtained for the three different DNA sequences are almost identical. The extent of contact with the protein was monitored for every base in the complex. Two concentration-dependent cleavage sites on the complementary TTCC strand are evidence of a specific interaction between PU.1 and the DNA. Two more protection sites and a hypersensitive cleavage site on the GGAA strand were observed. Although these data confirm the global structure of the PU.1-DNA complex as suggested by crystallography, the footprinting data reveal differences between the protein-DNA contacts in solution and in the crystal state. An additional interaction site not present in the crystal structure was observed by hydroxyl radical footprinting.

Animals↗

PubMed2005

Gene duplication and complex circadian clocks in mammals.

The circadian clock arose early in the evolution of life to enable organisms to adapt to the cycle of day and night. Recently, the extent and importance of circadian regulation of behaviour and physiology has come to be more fully realized. Core molecular cogs of circadian oscillators appear to have been largely conserved between such diverse organisms as Drosophila melanogaster and mammals. However, gene duplication events have produced multiple copies of many clock genes in mammals. Recent studies suggest that genome duplication has lead to increased circadian complexity and local tissue regulation. This has important implications for temporal regulation of behaviour via multiple clocks in the central nervous system, and also extends to the local physiology of major body organs and tissues.

Animals↗

PubMed2002

Donor strand complementation mechanism in the biogenesis of non-pilus systems.

The F1 antigen of Yersinia pestis belongs to a class of non-pilus adhesins assembled via a classical chaperone-usher pathway. Such pathways consist of PapD-like chaperones that bind subunits and pilot them to the outer membrane usher, where they are assembled into surface structures. In a recombinant Escherichia coli model system, chaperone-subunit (Caf1M:Caf1n) complexes accumulate in the periplasm. Three independent methods showed that these complexes are rod- or coil-shaped linear arrays of Caf1 subunits capped at one end by a single copy of Caf1M chaperone. Deletion and point mutagenesis identified an N-terminal donor strand region of Caf1 that was essential for polymerization in vitro, in the periplasm and at the cell surface, but not for chaperone-subunit interaction. Partial protease digestion of periplasmic complexes revealed that this region becomes buried upon formation of Caf1:Caf1 contacts. These results show that, despite the capsule-like appearance of F1 antigen, the basic structure is assembled as a linear array of subunits held together by intersubunit donor strand complementation. This example shows that strikingly different architectures can be achieved by the same general principle of donor strand complementation and suggests that a similar basic polymer organization will be shared by all surface structures assembled by classical chaperone-usher pathways.

Amino Acid Sequence↗