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Human immunodeficiency virus type 1 (HIV-1) and other lentiviridae demonstrate a strong preference for the A-nucleotide, which can account for up to 40% of the viral RNA genome. The biological mechanism responsible for this nucleotide bias is currently unknown. The increased A-content of these viral genomes corresponds to the typical use of synonymous codons by all members of the lentiviral family (HIV, SIV, BIV, FIV, CAEV, EIAV, visna) and the human spuma retrovirus, but not by other retroviruses like the human T-cell leukemia viruses HTLV-1 and HTLV-II. In this article, we analyzed A-bias for all codon groups in all open reading frames of several lentiviruses. The extent of lentiviral codon bias could be related to host cellular translation. By calculating codon bias indices (CBIs), we were able to demonstrate an inverse correlation between the extent of codon bias and the rate of translation of individual reading frames in these viruses. Specifically, the shift toward A-rich codons is more pronounced in pol than in gag lentiviral genes. Since it is known that Gag synthesis exceeds Pol synthesis by a factor of 20 due to infrequent ribosomal frame-shifting during translation of the gap-pol mRNA molecule, we propose that the aminoacyl-tRNA availability in the host cell restricts the lentiviral preference for A-rich codons. In addition, less A-nucleotides were found in regions of the viral genome encoding multiple functions; e.g., overlapping reading frames (tat-rev-env) or in genes that overlap regulatory sequences (nef-LTR region).(ABSTRACT TRUNCATED AT 250 WORDS)
J Mol Evol 1995 Aug
PMID:The tendency of lentiviral open reading frames to become A-rich: constraints imposed by viral genome organization and cellular tRNA availability. 766 42

The fungal phytopathogen Magnaporthe grisea parasitizes a wide variety of gramineous hosts. In the course of investigating the genetic relationship between pathogen genotype and host specificity we identified a retroelement that is present in some strains of M. grisea that infect finger millet and goosegrass (members of the plant genus Eleusine). The element, designated grasshopper (grh), is present in multiple copies and dispersed throughout the genome. DNA sequence analysis showed that grasshopper contains 198 base pair direct, long terminal repeats (LTRs) with features characteristic of retroviral and retrotransposon LTRs. Within the element we identified an open reading frame with sequences homologous to the reverse transcriptase, RNaseH, and integrase domains of retroelement pol genes. Comparison of the open reading frame with sequences from other retroelements showed that grh is related to the gypsy family of retrotransposons. Comparisons of the distribution of the grasshopper element with other dispersed repeated DNA sequences in M. grisea indicated that grasshopper was present in a broadly dispersed subgroup of Eleusine pathogens, suggesting that the element was acquired subsequent to the evolution of this host-specific form. We present arguments that the amplification of different retroelements within populations of M. grisea is a consequence of the clonal organization of the fungal populations.
Mol Plant Microbe Interact
PMID:Grasshopper, a long terminal repeat (LTR) retroelement in the phytopathogenic fungus Magnaporthe grisea. 767 35

Eight new examples of retrotransposons of the Gypsy/Ty3 class have been identified in marine species. A 525-nt pol gene-coding region was amplified using degenerate primers from highly conserved regions and has extended the range of recognition of Gypsy/Ty3 far beyond those previously known. The following matrix shows the percentage AA divergence of the translations of this segment of the pol gene coding region. [table: see text] The underlines separate three groups of retrotransposons that can be recognized on the basis of this amino acid sequence. The new upper group shows surprising amino acid sequence similarity among members from the DNA of herring, sea urchin, starfish, and a tunicate. For example, the herring element differs by only 41% from the Ciona element and 46% from the sea urchin element. The group between the lines includes members close to previously known elements (marked by asterisks) and has so far been found only in sea urchins. The two upper groups differ from each other by 55-60% and yet members of both groups (e.g., Spr1 and Spr2) are integrated into the DNA of one species--S. purpuratus. Below the lower underline is listed the only known representative of a very distant group, which occurs in starfish DNA. In spite of large divergence, amino acid sequence comparisons indicate that all of the elements shown in the array are members of the LTR-containing class of retrotransposons that includes Gypsy of Drosophila and Ty3 of yeast. Of all known mobile elements this class shows the closest sequence similarity to retroviruses and has the same arrangement of genes as simpler retroviruses.
J Mol Evol 1995 Jan
PMID:Gypsy/Ty3-class retrotransposons integrated in the DNA of herring, tunicate, and echinoderms. 771 10

To identify regions of the mitochondrial genome from the polima or pol male-sterile cytoplasm of Brassica napus that are genetically correlated with cytoplasmic male sterility (CMS) we analyzed mtDNAs of three male-sterile somatic hybrids formed by the fusion of broccoli (B. oleracea L. var. italica) and pol CMS B. napus protoplasts. Fragments characteristic of a 4.5 kb DNA segment that comprises the single organizational difference between sterile pol and fertile cam Brassica mitochondrial genomes were found in all three sterile somatic hybrids. One of these hybrids possessed a mitochondrial genome that was, apart from a limited region around this 4.5 kb CMS-associated segment, collinear with B. oleracea mtDNA. Previous studies have indicated that expression of transcripts spanning the atp6 gene and a chimeric gene, orf24, located on this 4.5 kb DNA segment, is associated with male sterility. The present results indicate that the orf224/atp6 gene region is genetically correlated with male sterility and provide significant additional support for the view that this gene region may be involved in specifying the CMS trait.
Plant Mol Biol 1995 Feb
PMID:Genetic correlation of the orf224/atp6 gene region with Polima CMS in Brassica somatic hybrids. 772 56

The nearly one million Alu repetitive elements in the human genome can be grouped into a number of subfamilies. Comparisons between subfamily consensus sequences suggest that Alu evolution is characterized by the sequential amplification and dispersal of a limited number of Alu founder sequences. The S, Sb and Sb1 subfamilies provide an example of such a related series of Alu subfamilies. We have previously demonstrated that adenovirus type 5 and herpes simplex virus type 1 activate RNA polymerase III transcription of endogenous Alu elements in HeLa cells. Here, we report that expression of Alu sequences belonging to the S, Sb and Sb1 subfamilies was activated following infection with these viruses. The data indicate that transpositionally inactive Alu elements can give rise to high levels of pol III transcripts in the presence of appropriate trans-acting factors and demonstrate that the class III promoters of a significant number and variety of Alu sequences are functional in vivo. Multiple subfamilies of Alu sequences were induced in transformed and non-transformed cell types, suggesting that induction of Alu expression may be part of the normal cellular response to viral infection.
J Mol Biol 1995 May 05
PMID:Activation of expression of multiple subfamilies of human Alu elements by adenovirus type 5 and herpes simplex virus type 1. 775 21

Eukaryotic cellular mRNA is believed to be synthesized exclusively by RNA polymerase II (pol II), whereas pol I produces long rRNAs and pol III produces 5S rRNA, tRNA, and other small RNAs. To determine whether this functional differentiation is obligatory, we examined the translational potential of an artificial pol III transcript. The coding region of the human immunodeficiency virus type 1 tat gene was placed under the control of a strong pol III promoter from the adenovirus type 2 VA RNAI gene. The resultant chimera, pVA-Tat, was transcribed accurately in vivo and in vitro and gave rise to Tat protein, which transactivated a human immunodeficiency virus-driven chloramphenicol acetyltransferase reporter construct in transfected HeLa cells. pol III-specific mutations down-regulated VA-Tat RNA production in vivo and in vitro and dramatically reduced chloramphenicol acetyltransferase transactivation. As expected for a pol III transcript, VA-Tat RNA was not detectably capped at its 5' end or polyadenylated at its 3' end, but, like mRNA, it was associated with polysomes in a salt-stable manner. Mutational analysis of a short open reading frame upstream of the Tat-coding sequence implicates scanning in the initiation of VA-Tat RNA translation despite the absence of a cap. In comparison with tat mRNA generated by pol II, VA-Tat RNA was present on smaller polysomes and was apparently translated less efficiently, which is consistent with a relatively low initiation rate. Evidently, human cells are capable of utilizing pol III transcripts as functional mRNAs, and neither a cap nor a poly(A) tail is essential for translation, although they may be stimulatory. These findings raise the possibility that some cellular mRNAs are made by pol I or pol III.
Mol Cell Biol 1995 Jul
PMID:Functional mRNA can be generated by RNA polymerase III. 779 67

A short interspersed repeat (SINE) in the two sibling species Chironomus pallidivittatus and Chironomus tentans is described. It is present at many sites in the genome and is surrounded by 10 to 14 bp target site duplications. It consists of two sequence modules in different numbers and variable order relative to each other and often has large inversions of different sizes at one end. One of the modules contains pol III promoter consensus sequences. This SINE, nevertheless, is likely to have been dependent on an outside promoter for its formation. It is therefore interesting that both modules start with a 22 bp region with striking similarity to the R2 insertion site in the preribosomal gene of insects. We suggest that this type of SINE, termed Cp1, was formed after a series of events among which the first step was the retroposition of a tRNA gene into the R2 site in the preribosomal gene by the R2 coded protein. The final step is likely to have been due to retroposition from this site.
J Mol Biol 1995 Jan 06
PMID:Polymorphic SINEs in chironomids with DNA derived from the R2 insertion site. 782 18

We have constructed a series of reporter constructs which test the effects of sequence elements from the control region of human mitochondrial DNA on expression in the nucleus, as assayed by transient expression in cultured human cells. The mitochondrial heavy-strand promoter (HSP) was unable to function as a promoter in nuclear DNA. Neither the HSP, nor the binding region for the mitochondrial transcription factor mtTF1 from the light-strand promoter, had any significant or systematic modulatory effects upon transcription from strong or weak RNA polymerase II (pol II) promoters, in three different human cell lines. The same finding held true regardless of orientation with respect to the start site of transcription. Similar results were obtained with a rho 0 derivative of one of these lines, indicating that mitochondrial promoter sequences in the nucleus cannot modulate transcription in response to altered mtDNA copy number. These results support the view that the nuclear and mitochondrial transcription systems in human cells are functionally independent, and do not communicate through factors recognizing shared sequence elements, as suggested by studies in yeast.
Mol Gen Genet 1994 Dec 15
PMID:Apparent functional independence of the mitochondrial and nuclear transcription systems in cultured human cells. 783 Jul 24

We have examined the biophysical properties of DNA polymerase beta (beta-pol) in solution. Time-resolved and steady-state fluorescence were used to investigate the microenvironment of the lone tryptophanyl residue (Trp324), and a combination of sedimentation equilibrium, sedimentation velocity and fluorescence anisotropy decay measurements were used to study the hydrodynamic properties of the enzyme. Trp324 appears to be exposed to water as judged by the tryptophan emission and steady-state and lifetime quenching experiments. The fluorescence is easily quenched by a neutral quencher acrylamide (kq = 1.59 x 10(9)M-1S-1), and by a negatively charged ionic quencher, I- (kq = 1.60 x 10(9) M-1S-1), but not by a positively charged ionic quencher, Cs+ (kq = 0.2 x 10(9) M-1S-1). The fluorescence lifetime of beta-pol is best described by the sum of two exponentials with a longer lifetime component of 8.4 ns and a shorter lifetime component of 1.3 ns. Decay associated spectra (DAS) show emission maxima at 340 nm and at 345 nm for the shorter lifetime and longer lifetime components, respectively, with corresponding centers of gravity at 347 nm and 348 nm. Sedimentation equilibrium experiments show that the enzyme exists as a monomer at the KCl concentrations (> 0.05 M) studied in the absence of divalent metals. Zn2+ causes higher order aggregation, but no such aggregates are seen with Mg2+ and Mn2+. In the presence of 1 mM manganese, the average lifetime decreased approximately 10%, from 8.14 ns to 7.38 ns, with a concomitant increase of average rotational correlational time (phi) from 24 ns to 28 ns. The accessibility of the positively charged quencher (Cs+) to tryptophan also decreases approximately 50%, indicating alteration of the tryptophan microenvironment. By contrast, Mg2+ causes minor changes in fluorescence properties. The hydrodynamic shape of the intact enzyme and its single-stranded (8 kDa) and double-stranded (31 kDa) DNA binding domains were further investigated by sedimentation velocity measurements. The value of S0(20),W for the intact enzyme is 2.97 S, and the calculated axial ratio is 5.0. In contrast to the 8 kDa domain, which has a less asymmetric shape with an axial ratio of 2.3, the 31 kDa domain shows an elongated structure with an axial ratio of 5.5. These data suggest that the axial ratio of the intact enzyme may be the result of marked bending of the molecule at the flexible hinge region between the two domains.
J Mol Biol 1994 Nov 25
PMID:Characterization of the tryptophan fluorescence and hydrodynamic properties of rat DNA polymerase beta. 796 32

An in vitro transcription system from Candida utilis is described. The template used is a hybrid plasmid containing Saccharomyces cerevisiae CYC1 promoter linked to a synthetic 377-bp G-minus casette (1). In vitro transcriptions are carried out in the presence of RNase. T1. Under these conditions only the transcripts that are resistant to RNase T1 accumulate. Using this protocol, it has been shown that in the absence of cytosolic factors RNA polymerase II (pol II) from C. utilis initiated RNA synthesis randomly. But both C. utilis and S. cerevisiae cell-free extracts could direct pol II from C. utilis to initiate transcription accurately. Results also indicated that the general transcription factors are functionally interchangeable between S. cerevisiae and C. utilis.
Biochem Mol Biol Int 1994 Aug
PMID:Accurate transcription initiation by RNA polymerase II from Candida utilis. 798 59


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