Number Of Open Reading Frames Research Articles

Two Related Rolling Circle Replication Plasmids from Salt-Tolerant Bacteria

In a range of salt-tolerant bacteria isolated from soil, rhizosphere, and phyloplane, cross-hybridization tests revealed a group of seven plasmids which were chosen for further study. Two plasmids (pSH1418 and pSH1451) were picked as representative examples of the two related subgroups. Restriction mapping and Southern blotting identified a region common to the two plasmids which later was identified as encoding the replication functions. We were unable to join these plasmids to high-copy-number vectors but this was possible with low-copy-number IncP vectors. InEscherichia coli,cloned plasmids pSH1418 and pSH1451 conferred salt tolerance but the phenotype was unstable, with the loss of salt tolerance apparently being correlated with structural instability of the plasmid DNA. Plasmid pSH1451 was sequenced and shown to be closely related to RCR plasmids of Gram-positive bacteria. The host of this plasmid was classified asBacillus pumilusby rDNA typing and lipid profiling by gas chromatography. A number of open reading frames (orfs) which could code for salt tolerance or other functions were identified in the plasmid sequence. Sequence similarity to previously sequenced genes suggested that the products of orf4 and orf5 may work together to transport a molecule such as aspartate ion that may promote osmotolerance.

Physical mapping of the conjugative bacteriocin plasmid pPD1 of Enterococcus faecalis and identification of the determinant related to the pheromone response.

The pheromone-responding conjugative bacteriocin plasmid pPD1 (59 kb) of Enterococcus faecalis was mapped physically by using a relational clone approach, and transposon analysis with Tn917 (Emr) or Tn916 (Tcr) facilitated the location of the bacteriocin-related genes in a segment of about 6.7 kb. Tn917 insertions within a 3-kb region resulted in constitutive clumping. The nucleotide sequence of the region that included the insertions giving rise to constitutive clumping was determined. The region of pPD1 spanned about 8 kb and was found to contain a number of open reading frames, some of which were named on the basis of homologies with two other pheromone-responding plasmids, pAD1 and pCF10. The genes were arranged in the sequence repB-repA-traC-traB-traA-ipd-traE-traF- orfY-sea-1 with all but repB and traA oriented in the same (left-to-right) direction. traC and traB corresponded, respectively, to traC and traB of pAD1 and to prgY and prgZ of pCF10.

The vaccinia virus J5L open reading frame encodes a polypeptide expressed late during infection and required for viral multiplication

A number of open reading frames (ORFs) are found in the vaccinia virus (VV) genome whose activities in the viral life cycle have not yet been determined. This report examines one such ORF, designated J5L, which was demonstrated to be essential for viral multiplication. Stable inactivation of the J5L ORF by insertion of a lacZ ORF was impossible unless another copy of the J5L ORF was present in the VV genome. Fusion genes between the J5L ORF and either the lacZ gene or the VV K1L gene were employed to study its temporal expression as well as its protein product. These experiments showed that J5L is transcribed late in infection and gives rise to a protein product which migrates by SDS-PAGE with the expected molecular weight (16 kDa). Numerous unsuccessful attempts to establish a stable cell line expressing J5L suggest that the J5L gene product could be cytotoxic.

Initiation codons within 5′-leaders of mRNAs as regulators of translation

Why should a preponderance of proto-oncogenes, growth factor genes and growth factor receptor genes contain translation initiation codons and associated open reading frames in their 5'-leaders? An increasing number of open reading frames are being shown to function as cis-acting regulatory signals able to moderate expression of the downstream reading frame. These regulatory elements could play a fundamental role in the regulation of proliferation of vertebrate cells.

Rod structure of a phycoerythrin II-containing phycobilisome. I. Organization and sequence of the gene cluster encoding the major phycobiliprotein rod components in the genome of marine Synechococcus sp. WH8020.

Phycobilisomes of the unicellular marine cyanobacteria are unique in having rod substructures with two distinct phycoerythrins, PE I and PE II, with five and six bilins, respectively (Ong, L. J., and Glazer, A. N. (1991) J. Biol. Chem. 266, 9515-9527). The genes for the alpha and beta subunits of PE I, PE II, and phycocyanin, and that for the PE II-associated linker polypeptide, are clustered on a single 15-kilobase region of the genome of Synechococcus sp. WH8020. Complete sequencing of this region allowed definitive assignment of the positions of all bilin attachment sites in these phycobiliproteins. Twelve other open reading frames are closely associated with the structural genes specified above. Six are homologous to open reading frames adjacent to phycobiliprotein genes in other cyanobacteria and inferred to be involved in bilin addition. This is the largest number of open reading frames of this class known in any cyanobacterium. Another of the open reading frames has a short region of striking similarity to the active site sequence of a bovine protein-phosphotyrosine phosphatase.

Determination of the Position of the Boundaries of the Terminal Repetitive Sequences within the Genome of Molluscum Contagiosum Virus Type 1 by DNA Nucleotide Sequence Analysis

The repetitive DNA sequences of the genome of Molluscum contagiosum virus type 1 (MCV-1) have been localized within the terminal regions of the viral genome corresponding to the BamHI MCV-1 DNA fragments B (18 kbp; 0 to 0.096 map units (m.u.)) and E (10.5 kbp; 0.944 to 1 m.u.). The fine mapping of these particular regions of the genome of MCV-1 revealed that the boundaries of the terminal repetitive DNA sequences of the viral genome are located within the DNA sequences of the HindIII MCV-1 DNA fragments K (3.8 kbp; 0.014 to 0.036 m.u.) and J1 (4.1 kbp; 0.962 to 0.985 m.u.). The exact position of the boundary of the repetitive DNA sequences was determined by DNA nucleotide sequencing. The HindIII DNA fragments K and J1 compose 3859 and 4107 bp, respectively. The DNA sequences of HindIII MCV-1 DNA fragment K possess repetitive DNA sequences between the nucleotide positions 1 and 1675 which are homologous to the inverted and complementary DNA sequences of the Hin dIII MCV-1 DNA fragment J1 between the nuclotide positions 2437 and 4107 (1670 bp). The degree of DNA sequence homology detected between the repetitive DNA sequences in the HindIII DNA fragments K and J1 of the viral genome was found to be 98%. The number of open reading frames (ORFs) detected by the analysis of the DNA sequences of the HindIII MCV-1 DNA fragments K and J1 was found to be 14 (70 to 219 amino acid residues) and 11 (70 to 365 amino acid residues), respectively.

Myxoma virus expresses a secreted protein with homology to the tumor necrosis factor receptor gene family that contributes to viral virulence

Poxviruses are known to contain a large number of open reading frames, particularly near the termini of the viral genome, that are not required for growth in tissue culture. However, many of these these gene products are believed to play important roles in determining the virulence of the virus by modulating the host immune response to the infection. Recently it has been shown that Shope fibroma virus encodes, within the terminal inverted repeats, a protein (T2) related to the cellular tumor necrosis factor receptor (TNFR) and which specifically binds both TNFa and TNFβ We have sequenced the terminal regions of two other Leporipoxviruses (myxoma virus and malignant rabbit fibroma virus) that are extremely invasive and capable of inducing extensive immunosuppression in rabbits and demonstrate that they also encode a closely related T2 homolog with all the structural motifs predicted for a secreted TNF binding protein. To investigate the biological role of the T2 protein, we have inactivated the myxoma virus T2 gene within each copy of the viral TIR by the insertion of a dominant selectable marker ( Escherichia coli guanosine phosphoribosyltransferase) and selection of the recombinant virus in the presence of mycophenolic acid. The success of the inactivation of both copies of T2 was confirmed by the loss a broad protein band (52–56 kDa) of the predicted size for T2 from the profile of proteins secreted from mutant virus-infected BGMK cells at earlytimes after infection. Although the T2-minus recombinant myxoma virus grew normally in tissue culture, upon infection of susceptible rabbits the viral disease was observed to be significantly attenuated. The majority of infected rabbits were able to mount an effective immune response to the infection and completely recovered. These survivor rabbits became immune to subsequent challenge with wild type myxoma virus. We conclude that the T2 viral protein is an important secreted virulence factor and that it in all likelihood functions by compromising the antiviral effects of TNF. We propose the term “viroceptor” to describe viral-encoded homologs of cellular lymphokine receptors whose function is to intercept the activity of the cognate lymphokine in order to short circuit the host immune response to the viral infection.

The trypanosome VSG expression site encodes adenylate cyclase and a leucine-rich putative regulatory gene.

African trypanosomes are protozoan parasites that evade the host immune system by varying their dense antigenic coat. The Variant Surface Glycoprotein (VSG) is expressed exclusively from telomere-linked expression sites that contain in addition to the VSG gene, a number of open reading frames termed Expression Site Associated Genes (ESAGs). Here we demonstrate by complementation of a yeast mutant deleted for adenylate cyclase (cyr-1), that an ESAG from Trypanosoma equiperdum encodes an adenylate cyclase. Furthermore, we report that adjacent to adenylate cyclase in the expression site, is a separate open reading frame that encodes a protein sequence motif similar to the leucine-rich repeat regulatory domain of Saccharomyces cerevisiae and Schizosaccharomyces pombe adenylate cyclases. The finding of two adjacent open reading frames homologous to a single enzyme in yeast suggests that the two expression site encoded proteins may interact to regulate adenylate cyclase activity during the course of an infection.

Newly identified groups of genes in chloroplasts.

Abstract The complete nucleotide sequences of the chloroplast genomes of a liverwort ( Marchantia polymorpha ) and a higher plant ( Nicotiana tabacum ) have been determined. A comparison of these demonstrates that chloroplast gene number and organization has been highly conserved in evolution. More importantly, analysis of these sequences has resulted in the identification of a number of open reading frames which are likely to represent additional, previously unrecognized, chloroplast genes.

Expression of cauliflower mosaic virus reverse transcriptase in yeast

Cauliflower mosaic virus (CaMV) is a double-stranded DNA plant virus1–3 for which evidence has been presented that its replicative cycle involves a reverse transcription step4–14. Until now, however, there has been no direct evidence for such a step. The sequence of the CaMV genome contains a number of open reading frames, one of which (ORF V) encodes a protein of predicted sequence homologous to retroviral reverse transcriptases10. We have cloned this gene, and expressed it in the yeast Saccharomyces cerevisiae. We report here that yeast expressing this gene accumulate significant levels of reverse transcriptase activity. This provides strong evidence that CaMV really does replicate through an RNA intermediate, converted to DNA by reverse transcriptase.