Product Of Open Reading Frame Research Articles

Hepatitis C Virus Infection, Mixed Cryoglobulinemia, and Non-Hodgkin's Lymphoma: an Emerging Picture

Hepatitis C virus (HCV) is a single-stranded RNA agent which expresses its genetic informations in the form of a single, large polyprotein encoded by an open reading frame (ORF) that extends through most of its genomic RNA. Proteolytic cleavage of the ORF product is essential for the virogenesis and the production of viral progeny. HCV is responsible for chronic liver disease, cirrhosis and possibly hepatocellular carcinoma. Viral persistence is considered the greatest problem in the management of HCV infection. It may result from several mechanisms, two of which are established. In the first, the high rate of genetic variations during viral replication results in the production of mutants capable of escaping the immune attack. In the second, the virus infects cells of the immune system itself, which represent a privileged site that cannot be reached by virus-specific T cell response. Involvement of lymphoid cells in the early stages of HCV infection may provide insight into the pathobiologic patterns of extrahepatic dissemination (lymph nodes, major salivary glands, kidneys, blood vessels). Dissemination of HCV-infected lymphoid cells throughout the organism is likely to maintain a mobile and extensive reservoir of the virus. In this respect, extrahepatic sites may act as a source of continuous reinfection of hepatocytes. Studies of intrahepatic B lymphocytes indicate that they are infected with HCV, clonally expanded and activated to secrete IgM molecules with rheumatoid factor activity. This strongly suggests that HCV directly stimulates B cell expansion, which may result in an indolent stage of lymphoproliferation (i.e., mixed cryoglobulinemia) or in frank B cell non-Hodgkin's lymphoma (NHL). The frequency of NHL, however, is much lower than that of HCV infection, suggesting that HCV alone is not able to induce tumors and that cellular events, in addition to the presence of virus and virus-encoded products, are necessary in order to obtain a malignant B cell phenotype. The demonstration of HCV productive infection in bone marrow-recruited and circulating pluri-potent hematopoietic CD34+ stem cells indicates that HCV replication occurs in the early differentiation stages of hematopoietic progenitors. These are stable cell populations and are likely to represent the initial site of infection and a continuous source of virus production.

Expression, cloning, and characterization of a Candida albicans gene, ALA1, that confers adherence properties upon Saccharomyces cerevisiae for extracellular matrix proteins.

Adherence of Candida albicans to host tissues is a necessary step for maintenance of its commensal status and is likely a necessary step in the pathogenesis of candidiasis. The extracellular matrix (ECM) proteins are some of the host tissue and plasma proteins to which C. albicans adheres through adhesins located on the fungal cell surface. To isolate genes encoding ECM adhesins, an assay was developed based on the ability of yeast cells to adhere to magnetic beads coated with the ECM protein fibronectin, type IV collagen, or laminin. A C. albicans genomic library was constructed by cloning XbaI-partially-digested and size-selected fragments into pAUR112, an Escherichia coli-yeast low-copy-number shuttle vector. The C. albicans library was transformed into Saccharomyces cerevisiae YPH 499, and clones capable of adherence were selected by using ECM protein-coated magnetic beads. A plasmid containing an approximately 8-kb insert was isolated from 29 adherent clones. These clones exhibited adherence to all ECM protein-coated magnetic beads and to human buccal epithelial cells. The ALA1 gene (for agglutinin-like adhesin) was localized by subcloning it into a 5-kb XbaI fragment which retained the adherence phenotype in both orientations. The complete DNA sequence of the 5-kb insert was determined, and an open reading frame (ORF) encoding 1,419 amino acid residues was identified. Deletions from the 5' and 3' ends extending into the DNA sequence encoding the 1,419-amino-acid ORF product inactivated the adherence phenotype, suggesting that it is the coding region of the ALA1 gene. A database search identified ALA1 to be similar to the C. albicans ALS1 (for agglutinin-like sequence 1) protein and the S. cerevisiae agglutinin protein (AG alpha1), although the homology at the primary amino acid sequence level is limited to the first half of each of these proteins. ALA1 contains a central domain of six tandem repeats of 36 amino acids. We discuss the significance of various predicted ALA1 structural motifs and their relationships to function in the adherence process.

Expression of thiamin biosynthetic genes (thiCOGE) and production of symbiotic terminal oxidase cbb3 in Rhizobium etli.

In this paper we report the cloning and sequence analysis of four genes, located on plasmid pb, which are involved in the synthesis of thiamin in Rhizobium etli (thiC, thiO, thiG, and thiE). Two precursors, 4-methyl-5-(beta-hydroxyethyl)thiazole monophosphate and 4-amino-5-hydroxymethylpyrimidine pyrophosphate, are coupled to form thiamin monophosphate, which is then phosphorylated to make thiamin pyrophosphate. The first open reading frame (ORF) product, of 610 residues, has significant homology (69% identity) with the product of thiC from Escherichia coli, which is involved in the synthesis of hydroxymethylpyrimidine. The second ORF product, of 327 residues, is the product of a novel gene denoted thiO. A protein motif involved in flavin adenine dinucleotide binding was found in the amino-terminal part of ThiO; also, residues involved in the catalytic site of D-amino acid oxidases are conserved in ThiO, suggesting that it catalyzes the oxidative deamination of some intermediate of thiamin biosynthesis. The third ORF product, of 323 residues, has significant homology (38% identity) with ThiG from E. coli, which is involved in the synthesis of the thiazole. The fourth ORF product, of 204 residues, has significant homology (47% identity) with the product of thiE from E. coli, which is involved in the condensation of hydroxymethylpyrimidine and thiazole. Strain CFN037 is an R. etli mutant induced by a single Tn5mob insertion in the promoter region of the thiCOGE gene cluster. The Tn5mob insertion in CFN037 occurred within a 39-bp region which is highly conserved in all of the thiC promoters analyzed and promotes constitutive expression of thiC. Primer extension analysis showed that thiC transcription in strain CFN037 originates within the Tn5 element. Analysis of c-type protein content and expression of the fixNOQP operon, which codes for the symbiotic terminal oxidase cbb3, revealed that CFN037 produces the cbb3 terminal oxidase. These data show a direct relationship between expression of thiC and production of the cbb3 terminal oxidase. This is consistent with the proposition that a purine-related metabolite, 5-aminoimidazole-4-carboxamide ribonucleotide, is a negative effector of the production of the symbiotic terminal oxidase cbb3 in R. etli.

Ribonucleoprotein formation by the ORF1 protein of the non-LTR retrotransposon Tx1L in Xenopus oocytes.

The Tx1L elements constitute a family of site-specific non-LTR retrotransposons found in the genome of the frog Xenopus laevis . The elements have two open reading frames (ORFs) with homology to proteins of retroviruses and other retroelements. This study demonstrates an expected activity of one of the element-encoded proteins. The RNA binding properties of ORF1p, the product of the first ORF of Tx1L, were examined after expression from RNA injected into Xenopus oocytes. Using sucrose gradient sedimentation and non-denaturing gel electrophoresis, we show that ORF1p associates with RNA in cytoplasmic ribonucleoprotein (RNP) particles. Discrete RNPs are formed with well-defined mobilities. The ORF1p RNPs are distinct from endogenous RNPs that contain stored oocyte mRNAs and two specific endogenous mRNAs do not become associated with ORF1p. ORF1p appears to be capable of associating with its own mRNA and with other injected RNAs, independent of specific recognition sequences. Although nuclear localization of ORF1p was anticipated, based both on the supposed mechanism of transposition and on the presence of a potential nuclear localization signal, no significant fraction of the protein was found in the oocyte nucleus. Nonetheless, the RNA binding capability of ORF1p is consistent with the proposed model for transposition of non-LTR retrotransposons.

The sequence of palF, an environmental pH response gene in Aspergillus nidulans

To molecularly characterize the influence of external pH in the secretion of enzymes by filamentous fungi, we have cloned and sequenced the palF gene of Aspergillus nidulans ( An). An An wild-type (wt) chromosome VII-specific cosmid library was used to transform a palF15 mutant strain. Selection for complementation was done on medium containing β-glycerolphosphate as the sole P i source. Two cosmids were identified (W2G08 and W4G12) and further subcloning of one cosmid (W2G08) defined a 5-kb PstI genomic fragment, which fully complements the palF15 mutation. An internal fragment from the genomic clone recognized a single message of approx. 3.5 kb on Northern blot. cDNA clones were obtained from a λgt10 cDNA library and sequenced, showing a nucleotide (nt) sequence of 3311 base pairs (bp), with a 828 bp long 5′-untranslated region ( UTR). The major open reading frame (ORF) identified in the sequence codes for a putative 775 amino acid (aa) protein, which shares some similarity with two putative ORF products of Saccharomyces cerevisiae ( Sc).

Protease-deficient herpes simplex virus protects mice from lethal herpesvirus infection.

Null mutants and attenuated mutants of herpes simplex virus (HSV) have been shown to induce immunity against challenge from wild-type virus. Null viruses with a defect in late gene products would be expected to express more viral genes than viruses with defects in essential early gene products and thus induce a better immune response. Herpesviruses encode a late gene product (serine protease) that is autocatalytic and cleaves the capsid assembly protein during viral replication. To determine whether a virus with a mutation in this gene could induce immunity, we constructed a recombinant virus containing the gusA reporter gene in the protease domain of the HSV type 1 UL26 open reading frame (ORF). Consistent with previous results (M. Gao, L. Matusick-Kumar, W. Hurlburt, S. F. DiTusa, W. W. Newcomb, J. C. Brown, P. J. McCann, I. Deckman, and R. J. Colonno, J. Virol. 68:3702-3712, 1994), recombinant virus could be isolated only from helper cell lines expressing the product of the UL26 ORF. Mice inoculated with the recombinant virus were unaffected by doses of virus that were lethal to mice infected with wild-type virus. Mice which were previously inoculated with the recombinant virus were also protected by a subsequent challenge with wild-type virus in a dose-dependent manner. These results indicate that recombinant viruses lacking the protease gene are avirulent but render protection from subsequent challenge.

The UL2 Open Reading Frame of Bovine Herpesvirus 1 Encodes a Uracil‐DNA Glycosylase

Sequence analysis within the unique long segment of the bovine herpesvirus 1 (BHV-1) genome previously identified an open reading frame (ORF), designated UL2, whose deduced polypeptide of 204 amino acids contained a consensus uracil-DNA glycosylase (UDGase) signature sequence. To determine whether the BHV-1 UL2 ORF product has UDGase activity, we positioned the UL2 sequence down-stream of the T7 promoter on the vector pET-28b(+) and expressed it in Escherichia coli. Upon induction with isopropyl beta-D-thiogalactopyranoside these cells produced a 23-kDa protein, the molecular mass of which was in accordance with the prediction from the nucleotide sequence. A one-step purification procedure using nickel-chelating affinity chromatography resulted in a homogeneous preparation of this protein, which displayed specific UDGase activity in an in vitro enzyme assay. These results provide evidence that the BHV-1 UL2 gene does encode a UDGase.

Cloning and characterization of the genes encoding nitrilotriacetate monooxygenase of Chelatobacter heintzii ATCC 29600.

A 6.2-kb DNA fragment containing the genes for the nitrilotriacetate (NTA) monooxygenase of Chelatobacter heintzii ATCC 29600 was cloned and characterized by DNA sequencing and expression studies. The nucleotide sequence contained three major open reading frames (ORFs). Two of the ORFs, which were oriented divergently with an intergenic region of 307 bp, could be assigned to the NTA monooxygenase components A and B. The predicted N-terminal amino acid sequences of these ORFs were identical with those determined for the purified components. We therefore named these genes ntaA (for component A of NTA monooxygenase) and ntaB (for component B). The ntaA and ntaB genes could be expressed in Escherichia coli DH5alpha, and the gene products were visualized after Western blotting (immunoblotting) and incubation with polyclonal antibodies against component A or B. By mixing overproduced NtaB from E. coli and purified component A from C. heintzii ATCC 29600, reconstitution of a functional NTA monooxygenase complex was possible. The deduced gene product of ntaA showed only significant homology to SoxA (involved in dibenzothiophene degradation) and to SnaA (involved in pristamycin synthesis); that of ntaB shared weak homologies in one domain with other NADH:flavine mononucleotide oxidoreductases. These homologies provide no conclusive answer as to the possible evolutionary origin of the NTA monooxygenase. The deduced gene product of the third ORF (ORF1) had homology in the N-terminal region with the GntR class of bacterial regulator proteins and therefore may encode a regulator protein, possibly involved in regulation of ntaA and ntaB expression.

Characterization of IS199fromStreptococcus mutansV403

The complete nucleotide sequence of the insertion sequence IS199has been determined. This novel element was found inStreptococcus mutansV403 where DNA hybridization studies suggested that it was present in multiple copies on the genome. IS199is 1220 bp and appears unique to the mutans streptococci. A limited epidemiological survey revealed this element to occur infrequently in this group (4/50 strains examined). IS199belongs to the IS3family of procaryotic insertion sequence elements. It contained tandem open reading frames (ORFs) reminiscent of the structure of the IS3class. Additionally, the larger of the two ORF products (ORFB) exhibited amino acid sequence similarities to comparable proteins from IS3and other elements of this family. Transposition of IS199gave rise to a 3-bp target site duplication. We were able to insert an antibiotic resistance gene into IS199enabling us to follow the transposition of this element inS. mutansV403.

Subtelomeric duplications in Saccharomyces cerevisiae chromosomes III and XI: Topology, arrangements, corrections of sequence and strain-specific polymorphism

We have determined the sequence of a 3.42 kb segment from the left arm of chromosome III (coordinates 5394-8815 of Oliver et al., 1992). Instead of four open reading frames (ORFs) listed previously, the verified sequence reveals the presence of only one ORF, renamed YCL070/73c, encoding a protein of 615 amino acids. The putative product of ORF YCL070/73c shows 98.5% identity and 99% similarity with the protein of the same length encoded by ORF YKR106w from the right arm of chromosome XI and displays a topology characteristic for the Major Facilitators Superfamily of membrane proteins. These corrections will be deposited in the EMBL data library under the Accession Number X59720. In strain S288C the subtelomeric sequence 4319-11 215 of chromosome III is 98.3% identical with the subtelomeric sequence of 658 204-665 061 from the right arm of chromosome XI. Using various subtelomeric probes from chromosome III (coordinates 2097-3646 of S288C) we have analysed eight different Saccharomyces cerevisiae strains and the closely related species S. douglasii: some S. cerevisiae strains have additional duplications and longer chromosomes XI; in all strains chromosome III contains the 1200-11 000 segment (strain FL100 is disomic) while S. douglasii does not show any hybridization in this region.

Subtelomeric Duplications in Saccharomyces cerevisiae Chromosomes III and XI: Topology, Arrangements, Corrections of Sequence and Strain‐Specific Polymorphism

We have determined the sequence of a 3·42 kb segment from the left arm of chromosome III (coordinates 5394–8815 of Oliver et al., 1992). Instead of four open reading frames (ORFs) listed previously, the verified sequence reveals the presence of only one ORF, renamed YCL070/73c, encoding a protein of 615 amino acids. The putative product of ORF YCL070/73c shows 98·5% identity and 99% similarity with the protein of the same length encoded by ORF YKR106w from the right arm of chromosome XI and displays a topology characteristic for the Major Facilitators Superfamily of membrane proteins. These corrections will be deposited in the EMBL data library under the Accession Number X59720. In strain S288C the subtelomeric sequence 4319–11 215 of chromosome III is 98·3% identical with the subtelomeric sequence of 658 204–665 061 from the right arm of chromosome XI. Using various subtelomeric probes from chromosome III (coordinates 2097–3646 of S288C) we have analysed eight different Saccharomyces cerevisiae strains and the closely related species S. douglasii: some S. cerevisiae strains have additional duplications and longer chromosomes XI; in all strains chromosome III contains the 1200–11 000 segment (strain FL100 is disomic) while S. douglasii does not show any hybridization in this region.

Expression of the Barley Stripe Mosaic Virus RNAβ “Triple Gene Block”

Genomic RNAβ of barley stripe mosaic virus (BSMV) contains four defined open reading frames (ORFs). These include the coat protein (βa) and a “triple gene block” consisting of the βb, βc, and βd ORFs that overlap one another. Two subgenomic β RNAs (sgRNAβ1 and sgRNAβ2) with sizes of 2.5 and 0.96 kb were identified in BSMV-infected protoplasts, and their transcription initiation sites were mapped to nucleotides 789 and 2327, respectively, of RNAβ by primer extension experiments. In a cell-free wheat germ translation system, genomic RNAβ served as a mRNA only for the 22-kDa coat protein, and sgRNAβ1 directed synthesis of only the 58-kDa βb protein. However, with sgRNAβ2, three proteins with sizes of 14, 17, and 23 kDa were synthesized. Both the 14- and the 23-kDa proteins were recognized by the βd antibodiesin vitroandin vivo.These results demonstrated that the 14-kDa protein was encoded by the βd ORF and suggested that the 23-kDa protein, designated βd′, is a readthrough product of the amber stop codon of the βd ORF. Mutagenesis of sgRNAβ2 revealed that the 17-kDa protein was a product of the βc ORF. Expression of sgRNAβ1 and sgRNAβ2 was also investigated with the chloramphenicol acetyl transferase (CAT) reporter gene in protoplasts coinfected with RNAs α and γ plus chimeric RNAβ derivatives containing the CAT gene in-frame with the βb, βc, βd, or βd′ ORFs. Elimination of the sgRNAβ1 promoter abolished CAT expression from the βb-CAT chimeric RNA, and removal of the sgRNAβ2 promoter prevented CAT expression from the βc-CAT, βd-CAT, and βd′-CAT chimeric RNAs. Taken together, these results demonstrate that the BSMV coat protein is the sole translation product of the genomic RNAβ, whereas sgRNAβ1 serves as a messenger for translation of the βb protein, and sgRNAβ2 functions as a messenger for translation of βc and βd and the newly discovered βd′ protein. Additional mutagenesis experiments indicate that βc is translated by a leaky scanning mechanism.

Cloning and nucleotide sequence of a xylanase-encoding gene from Streptomyces sp. strain EC3.

Using the p1J702 vector, a xylanase-encoding gene (xin) of Streptomyces sp. EC3 has been cloned by functional complementation of a mutant of Streptomyces lividans TK24, producing xylanase at a very low level. Normal level of xylanase synthesis was restored in at least three clones, containing the same 3802 bp Sstl DNA fragment. In this fragment, several open reading frames (ORFs) have been identified, one of which coded for a xylanase; the products of the other ORFs did not show homology with any of the already known proteins. The complete nucleotide sequence of the 3802 bp Ssti insert has been determined on both strands. Xylanase is very probably synthesized as a 240 amino acid (aa) precursor (25949 Da) including a long (49 aa) signal sequence presenting significant similarity with the signal sequences of other Streptomyces xylanase genes. The xylanase aa sequence showed a clear homology with the aa sequences of other xylanases of the glycanase G family. The xln gene has been introduced into Streptomyces parvulus, a naturally xylanase-negative species. In contrast with its expression in Streptomyces sp. EC3, in S. parvulus, xln was expressed constitutively, a probable consequence of the absence of a regulatory system.

Cloning and characterization of the Bacillus subtilis prkA gene encoding a novel serine protein kinase

We have cloned and sequenced a 3574-bp Bacillus subtilis (Bs) DNA fragment located between the nrdA and citB genes at about 169° on the chromosome. An Escherichia coli strain, LBG1605, carrying a mutated ptsH gene (encoding HPr (His-containing protein) of the bacterial phosphotransferase system (PTS)) and complemented for PTS activity with the ptsH of Staphylococcus carnosus, exhibited reduced mannitol fermentation activity when transformed with a plasmid bearing this 3574-bp Bs fragment. This fragment contained an incomplete and two complete open reading frames (ORFs). The product of the first complete ORF, a protein composed of 235 amino acids (aa) (25 038 Da), was found to be responsible for the observed reduced mannitol fermentation. The 3′ part of this 705-bp second ORF and the 428-bp incomplete first ORF encode aa sequences exhibiting almost 40% sequence identity. However, the function of these two proteins remains unknown. The third ORF, the 1893-bp prkA gene, encodes a protein (PrkA) of 72 889 Da. PrkA possesses the A-motif of nucleotide-binding proteins and exhibits distant homology to eukaryotic protein kinases. Several of the essential aa in the loops known to form the active site of cyclic adenosine 3′,5′-monophosphate (cAMP)-dependent protein kinase appeared to be conserved in PrkA. After expression of prkA and purification of PrkA, we could demonstrate that PrkA can indeed phosphorylate a Bs 60-kDa protein at a Ser residue.

The Epstein-Barr virus (EBV) BZLF2 gene product associates with the gH and gL homologs of EBV and carries an epitope critical to infection of B cells but not of epithelial cells

Glycoprotein gp85, the product of the BXLF2 open reading frame (ORF), is the gH homolog of Epstein-Barr virus (EBV) and has been implicated in penetration of virus into B cells. Like its counterparts in other herpesviruses, it associates with a gL homolog, gp25, which is the product of the BKRF2 ORF. Unlike the gH homologs of other herpesviruses, however, gp85 also complexes with two additional glycoproteins of 42 and 38 kDa. Glycoproteins gp42 and gp38 were determined to be alternatively processed forms of the BZLF2 gene product. Coexpression of EBV gH and gL facilitated transport of gH to the cell surface and resulted in formation of a stable complex of gH and gL. It also restored expression of an epitope recognized by monoclonal antibody E1D1, which immunoprecipitates the native gH complex but not recombinant gH expressed in isolation. Coexpression of gH, gL, and the BZLF2 ORF restored expression of an epitope recognized by a second monoclonal antibody, F-2-1, which immunoprecipitates the native gH-gL-gp42/38 complex but not the complex of recombinant gH and gL alone. The epitope recognized by antibody F-2-1 was mapped to the BZLF2 gene product itself. Antibody F-2-1 inhibited the ability of EBV to infect B lymphocytes but had no effect on the ability of the virus to infect the epithelial cell line SVK-CR2. In contrast, antibody E1D1 had no effect on infection of the B-cell line but inhibited infection of the epithelial cell line. These results indicate that penetration of the two cell types by EBV involves differential use of the gH-gL-gp42/38 complex and suggest the hypothesis that the BZLF2 gene product has evolved as a unique adaptation to infection of B lymphocytes by EBV.

The transcriptional regulatory proteins encoded by varicella-zoster virus open reading frames (ORFs) 4 and 63, but not ORF 61, are associated with purified virus particles.

Of the five varicella-zoster virus (VZV) open reading frames (ORFs) known to encode proteins which influence viral transcriptional events, two (ORFs 10 and 62) encode proteins associated with the tegument of virus particles, where they may function during the immediate-early events of infection. In this study, antibodies which recognize the products of the three additional VZV ORFs, ORFs 4, 61, and 63, were made and used to characterize their association with virus particles. ORF 4 encoded a 52-kDa polypeptide, and antibodies to ORF 63 reacted with polypeptides of 47 and 28 kDa. Antibodies to ORF 61 recognized heterogeneous polypeptides of 62 to 66 kDa in cells infected with a vaccinia virus recombinant expressing ORF 61 and in VZV-infected melanoma cells but reacted very weakly with polypeptides of VZV-infected human foreskin fibroblasts, suggesting that cell-specific factors were involved in ORF 61 protein accumulation. Analysis of virus particles purified from melanoma cells indicated that a 52-kDa polypeptide from ORF 4 and the 47-kDa polypeptide from ORF 63, but not any from ORF 61, were associated with virus particles. The virion proteins were likely components of the tegument, as they were not solubilized by treatment of virus with mild detergents and were completely resistant to trypsin digestion unless prior envelope solubilization was performed. The products of ORFs 4 and 63 were not found in purified VZV nucleocapsids. These results suggest that forms of the ORF 4- and ORF 63-encoded transcriptional regulatory proteins are also structural and may also have roles in the immediate-early events of infection.

Immediate-early transcription from the channel catfish virus genome: characterization of two immediate-early transcripts.

With cDNA probes and by Northern (RNA) blot analysis, a region containing immediate-early (IE) genes in the channel catfish virus (CCV) genome was identified. IE transcription in CCV-infected cells appears to be restricted to the terminal repeat region, suggesting that CCV is most closely related to the alpha subfamily of herpesviruses. CCV DNA fragments from this region encoding IE transcripts were cloned. Northern analysis with one of these cloned fragments, a 3,927-bp EcoRI-XbaI fragment, indicates that it encodes two IE transcripts. Both transcripts (ie1 and ie2) were characterized by S1 nuclease analysis, primer extension analysis, and analysis of cDNAs. The ie2 transcript is a 1.3-kb bicistronic mRNA containing open reading frame (ORF) 8a and ORF 9. ORF 8a is a 5'-truncated version of ORF 8 which, along with ORF 9, was previously identified (A. J. Davison, Virology 186:9-14, 1992). The ie1 transcript is 0.6 kb in size, contains only ORF 9, and is expressed at a level approximately six times that of ie2 in cycloheximide-treated cells. The putative product of ORF 9 is predicted to have a basic pI and contains a potential zinc-binding domain, making it a probable transcription factor. ORF 8a encodes a putative product which is very hydrophobic, an unusual characteristic for an IE protein.

Isolation and characterization of a novel mitogenic regulatory gene, 322, which is transcriptionally suppressed in cells transformed by src and ras.

In an attempt to isolate novel regulatory and/or tumor suppressor genes, we identified cDNAs whose abundance is low in NIH 3T3 cells and further decreased following the expression of the activated oncogene, v-src. The transcription of one such gene, 322, is suppressed at least 15-fold in src-, ras-, and fos-transformed cells and 3-fold in myc-transformed cells but is unaffected in raf-, mos-, or neu-transformed cells. Activation of a ts-v-src allele in confluent 3Y1 fibroblasts resulted in an initial increase in 322 mRNA levels after 1 to 2 h followed by a rapid decrease to suppressed levels after 4 to 8 h. Morphological transformation was not detected until 12 h later, indicating that the accumulation of 322 transcripts is regulated by v-src and not as a consequence of transformation. Addition of fetal calf serum to starved subconfluent NIH 3T3 or 3Y1 fibroblasts resulted in a similar biphasic regulation of 322, indicating that 322 transcription is responsive to mitogenic factors. Sequence analysis of a putative full-length 322 cDNA clone (5.4 kb) identified a large open reading frame (ORF) encoding a 148.1-kDa product. In vitro transcription and translation of the 322 cDNA from a T7 promoter resulted in a 207-kDa product whose electrophoretic mobility on a sodium dodecyl sulfate-polyacrylamide gel electrophoresis gel was unaffected by digestion with endoglycosidase F. The discrepancy in predicted versus measured molecular weights may result from the high percentage of acidic residues (roughly 20% Glu or Asp) in the 322 ORF product. Comparison of the 322 cDNA ORF with sequences in data banks indicates that this gene is novel. The 322 ORF product contains a potential Cys-1-His-3 Zn finger, at least five nuclear localization signals of the adenovirus E1a motif K(R/K)X(R/K), and alternating acidic and basic domains. Overexpression of the 322 cells resulted in the selection of rapidly growing cells which had lost the transduced 322 cDNA. Thus, 322 represent a novel src- and ras-regulated gene which encodes a potential regulator of mitogenesis and/or tumor suppressor.

The complete nucleotide sequence of RNA 3 of citrus leaf rugose and citrus variegation ilarviruses.

Complete sequence data for the RNA 3 of both citrus leaf rugose (CiLRV) and citrus variegation (CVV) ilarviruses have been determined. The RNAs are 2289 nt (CiLRV) and 2309 nt (CVV) in length and both contain the typical Bromoviridae arrangement of two open reading frames (ORFs) which, when translated, code for proteins that correspond to the Mr 32,000 (32K) putative movement proteins (ORF 1) and the coat proteins (ORF 2) of the respective viruses. The 3' termini of both viruses can be folded to form a secondary structure similar to those reported for other ilarviruses. These are the first complete nucleotide sequences for RNA 3 of members of subgroup 2 of the ilarviruses. The two viruses share substantial homology in nucleic acid sequence, code for identically sized coat proteins and share high levels of identity in the translated products of both ORFs. Although related, these viruses differ sufficiently to be considered distinct. The RNA 3s of CiLRV and CVV appear to be distinct from those of other ilarviruses for which comparable sequence data are available and also from the closely related alfalfa mosaic virus.

A gene required for nutritional repression of the Bacillus subtilis dipeptide permease operon

An insertion mutation was isolated that resulted in derepressed expression of the Bacillus subtillis dipeptide transport operon (dpp) during the exponential growth phase in rich medium. DNA flanking the site of insertion was found to encode an operon (codVWXY) of four potential open reading frames (ORFs). The deduced product of the codV ORF is similar to members of the lambda Int family; CodW and CodX are homologous to HsIV and HsIU, two putative heat-shock proteins from Escherichia coli, and to LapC and LapA, two gene products of unknown function from Pasteurella haemolytica. CodX also shares homology with a family of ATPases, including ClpX, a regulatory subunit of the E. coli ClpP protease. CodY does not have any homologues in the data-bases. The insertion mutation and all previously isolated spontaneous cod mutations were found to map in codY. In-frame deletion mutations in each of the other cod genes revealed that only codY is required for repression of dpp in nutrient-rich medium. The codY mutations partially relieved amino acid repression of the histidine utilization (hut) operon but had no effect on regulation of certain other early stationary phase-induced genes, such as spoVG and gsiA.