Identification of an operon involved in sulfolipid biosynthesis in Rhodobacter sphaeroides.

Two new mutants of Rhodobacter sphaeroides deficient in sulfolipid accumulation were isolated by directly screening mutagenized cell lines for polar lipid composition by thin-layer chromatography of lipid extracts. A genomic clone which complemented the mutations in these two lines, but not the previously described sulfolipid-deficient sqdA mutant, was identified. Sequence analysis of the relevant region of the clone revealed three, in tandem open reading frames, designated sqdB, ORF2, and sqdC. One of the mutants was complemented by the sqdB gene, and the other was complemented by the sqdC gene. Insertional inactivation of sqdB also inactivated sqdC, indicating that sqdB and sqdC are cotranscribed. The N-terminal region of the 46-kDa putative protein encoded by the sqdB gene showed slight homology to UDP-glucose epimerase from various organisms. The 30-kDa putative protein encoded by ORF2 showed very striking homology to rabbit muscle glycogenin, a UDP-glucose utilizing, autoglycosylating glycosyltransferase. The 26-kDa putative protein encoded by the sqdC gene was not homologous to any protein of known function.     

Cloning and sequencing of the gene encoding a 125-kilodalton surface-layer protein from Bacillus sphaericus 2362 and of a related cryptic gene.

Using the vector pGEM-4-blue, a 4,251-base-pair DNA fragment containing the gene for the surface (S)-layer protein of Bacillus sphaericus 2362 was cloned into Escherichia coli. Determination of the nucleotide sequence indicated an open reading frame (ORF) coding for a protein of 1,176 amino acids with a molecular size of 125 kilodaltons (kDa). A protein of this size which reacted with antibody to the 122-kDa S-layer protein of B. sphaericus was detected in cells of E. coli containing the recombinant plasmid. Analysis of the deduced amino acid sequence indicated a highly hydrophobic N-terminal region which had the characteristics of a leader peptide. The first amino acid of the N-terminal sequence of the 122-kDa S-layer protein followed the predicted cleavage site of the leader peptide in the 125-kDa protein. A sequence characteristic of promoters expressed during vegetative growth was found within a 177-base-pair region upstream from the ORF coding for the 125-kDa protein. This putative promoter may account for the expression of this gene during the vegetative growth of B. sphaericus and E. coli. The gene for the 125-kDa protein was followed by an inverted repeat characteristic of terminators. Downstream from this gene (11.2 kilobases) was an ORF coding for a putative 80-kDa protein having a high sequence similarity to the 125-kDa protein. Evidence was presented indicating that this gene is cryptic.     

Cloning and characterization of two immunophilin-like genes, ilpA and fkpA, on a single 3.9-kilobase fragment of Aeromonas hydrophila genomic DNA.

Antiserum to Aeromonas hydrophila A6 cell envelopes was shown in a previous study (C. Y. F. Wong, G. Mayrhofer, M. W. Heuzenroeder, H. M. Atkinson, D. M. Quinn, and R. L. P. Flower, FEMS Immunol. Med. Microbiol. 15:233-241, 1996) to protect mice against lethal infection by this organism. In this study, colony blot analysis of an A. hydrophila genomic library using antiserum to A. hydrophila A6 cell envelopes revealed a cosmid clone expressing a 30-kDa protein which has not been described previously in aeromonads. The nucleotide sequence of a 3.9-kb fragment derived from this cosmid which expressed the 30-kDa protein revealed two potential open reading frames (ORFs) with homology to known immunophilin proteins. ORF1 encoded a 212-amino-acid protein (molecular mass, 22.4 kDa) with 56% identity to the immunophilin SlyD protein of Escherichia coli. ORF1 was subsequently designated ilpA (immunophilin-like protein). ORF3 encoded a potential gene product of 268 amino acids with a typical signal sequence and a predicted molecular size of 28.7 kDa. The inferred amino acid sequence showed 46% identity with the sequence of the FkpA protein of E. coli and 40% identity with the sequence of the macrophage infectivity potentiator (Mip) protein of Legionella pneumophila. ORF3 was designated fkpA (FK506 binding protein) by analogy with the E. coli FkpA protein. Expression of the FkpA protein was confirmed by Western blot (immunoblot) analysis, which detected a 30-kDa protein, with antiserum to the Mip protein of Legionella longbeachae and a specific antiserum to anA. hydrophila 30-kDa membrane protein. PCR and Southern analysis showed that a DNA sequence encoding FkpA was found in all 178 aeromonads of diverse origins tested. A nonpolar insertion mutation in the fkpA gene did not attenuate virulence in a suckling mouse model nor did it affect the expression of hemolysins or DNase. This suggests that either the fkpA gene is not essential in the virulence of A. hydrophila under these conditions or there are other genes in A. hydrophila coding for proteins with similar functions.     

Varicella-Zoster Virus (VZV) ORF32 Encodes a Phosphoprotein That Is Posttranslationally Modified by the VZV ORF47 Protein Kinase

Varicella-zoster virus (VZV) encodes five gene products that do not have homologs in herpes simplex virus. One of these genes, VZV open reading frame 32 (ORF32), is predicted to encode a protein of 16 kDa. VZV ORF32 protein was shown to be phosphorylated and located in the cytosol of virus-infected cells. Antibody to ORF32 protein immunoprecipitated 16- and 18-kDa phosphoproteins from VZV-infected cells. Since VZV encodes two protein kinases that might phosphorylate ORF32 protein, immunoprecipitations were performed with cells infected with VZV mutants unable to express either of the viral protein kinases. Cells infected with VZV unable to express the ORF66 protein kinase contained both the 16- and 18-kDa ORF32 phosphoproteins; however, cells infected with the VZV ORF47 protein kinase mutant showed only the 16-kDa ORF32 phosphoprotein. Treatment of [³⁵S]methionine-labeled proteins with calf intestine alkaline phosphatase resulted in a decrease in size of the ORF32 proteins from 16 and 18 kDa to 15 and 17 kDa, respectively. VZV unable to express ORF32 protein replicated in human melanoma cells to titers similar to those seen with parental virus; however, VZV unable to express ORF32 was impaired for replication in U20S osteosarcoma cells. Thus, VZV ORF32 protein is posttranslationally modified by the ORF47 protein kinase. Since the VZV ORF47 protein kinase has recently been shown to be critical for replication in human fetal skin and lymphocytes, its ability to modify the ORF32 protein suggests that the latter protein may have a role for VZV replication in human tissues.     

Maize tapetum xylanase is synthesized as a precursor,processed and activated by a serine protease,and deposited on the pollen

Pollen coat contains ingredients that interact with the stigma surface during sexual reproduction. In maize (Zea mays L.) pollen coat, the predominant protein is a 35-kDa endoxylanase, whose mRNA is located in the tapetum cells enclosing the maturing pollen in the anthers. This 2.0-kb mRNA was found to have an open reading frame of 1,635 nucleotides encoding a 60-kDa pre-xylanase. In developing anthers, the pre-xylanase protein appeared prior to the 35-kDa xylanase protein and enzyme activity and then peaked and declined, whereas the 35-kDa xylanase protein and activity continued to increase until anther maturation. An acid protease in the anther extract converted the inactive pre-xylanase to the active 35-kDa xylanase in vitro. The protease activity was inhibited by inhibitors of serine proteases but unaffected by inhibitors of cysteine, aspartic, or metallic proteases. Sequence analysis revealed that the 60-kDa pre-xylanase was converted to the 35-kDa xylanase with the removal of 198 and 48 residues from the N and C termini, respectively. During in vitro and in vivo conversions, no intermediates of 60-35 kDa were observed, and the 35-kDa xylanase was highly stable. The pre-xylanase was localized in the tapetum-containing anther wall, whereas the 35-kDa xylanase was found in the pollen coat. The significance of having a large non-active pre-xylanase and the mode of transfer of the xylanase to the pollen coat are discussed. A gene encoding the barley (Hordeum vulgare L.) tapetum xylanase was cloned; this gene and the gene encoding the seed aleurone-layer xylanase had strict tissue-specific expressions.     

Characterization of the human 36-kDa carboxyl terminal LIM domain protein (hCLIM1)

We characterized a human cDNA clone encoding a 36-kDa carboxyl terminal LIM domain protein with a PDZ domain at the amino terminal. This full-length cDNA clone has a predicted open reading frame (ORF) of 329 amino-acid residues. The ORF of this cDNA encodes the human homolog of rat CLP36, and the putative protein is named human 36-kDa carboxyl terminal LIM domain protein (hCLIM1, nomenclature approved by the HUGO/GDB Nomenclature Committee). The hCLIM1 probe was used to hybridize with poly(A)+ RNA of various human tissues. Strong signals were detected in heart and skeletal muscle; moderate signals were detected in spleen, small intestine, colon, placenta, and lung; weaker levels were detected in liver, thymus, kidney, prostate, and pancreas; and no observable signals were detected in brain, testis, ovary, and peripheral blood leukocytes. The hCLIM1 gene was studied by fluorescence in situ hybridization (FISH), somatic cell hybrid analysis, and radiation hybrid mapping, and it is located at the human chromosome 10q26.     

The gene encoding the 17-kDa antigen of Bartonella henselae is located within a cluster of genes homologous to the virB virulence operon

A Bartonella henselae genomic A library was screened with antiserum generated in mice against live B. henselae. One of the immunoreactive clones expressed a 17-kDa antigen that was characterized previously as an immunodominant protein of B. henselae. Sequence analysis of the recombinant clone, pBHIM-2, revealed that the open reading frame (ORF) encoding the 17-kDa antigen was situated between homologs of virB4 and virB6, two genes that belong to the virB operon. The virB operon has been associated with the transfer of oncogenic T-DNA in Agrobacterium tumefaciens and with secretion of the pertussis toxin in Bordetella pertussis. Downstream of the virB6 gene within pBHIM-2 was a partial open reading frame that was homologous to the virB8 gene. Rescreening of the library by plaque hybridization using probes specific to the 5' and 3' ends of the pBHIM-2 insert resulted in the isolation of recombinant clones containing additional virB genes. Assembly of the sequences obtained from the recombinant clones revealed that eight of the open reading frames encode homologs of the VirB proteins. The homology and colinearity with the virB genes suggest that the gene encoding the 17-kDa antigen is expressed within the virB locus of B. henselae.     

Identification and characterization of the herpes simplex virus type 1 virion protein encoded by the UL35 open reading frame.

The UL35 open reading frame (ORF) of herpes simplex virus type 1 (HSV-1) has been predicted from DNA sequence analysis to encode a small polypeptide with a molecular weight of 12,095. We have investigated the protein product of the UL35 ORF by using a trpE-UL35 gene fusion to produce a corresponding fusion protein in Escherichia coli. The TrpE-UL35 chimeric protein was subsequently isolated and used as a source of immunogen for the production of rabbit polyclonal antiserum directed against the UL35 gene product. The TrpE-UL35 antiserum was found to recognize a 12-kDa protein which was specifically present in HSV-1-infected cells. By utilizing the TrpE-UL35 antiserum, the kinetics of synthesis of the UL35 gene product was examined, and these studies indicate that UL35 is expressed as a gamma 2 (true late) gene. The 12-kDa protein recognized by the TrpE-UL35 antiserum was associated with purified HSV-1 virions and type A and B capsids, suggesting that the UL35 ORF may encode the 12-kDa capsid protein variably designated p12, NC7, or VP26. To confirm this assignment, immunoprecipitation and immunoblotting studies were performed to demonstrate that the TrpE-UL35 antiserum reacts with the same polypeptide as an antiserum directed against the purified p12 capsid protein (anti-NC7) (G.H. Cohen, M. Ponce de Leon, H. Diggelmann, W.C. Lawrence, S.K. Vernon, and R.J. Eisenberg, J. Virol. 34:521-531, 1980). Furthermore, the anti-NC7 serum was also found to react with the TrpE-UL35 chimeric protein isolated from E. coli, providing additional evidence that the UL35 gene encodes p12. On the basis of these studies, we conclude that UL35 represents a true late gene which encodes the 12-kDa capsid protein of HSV-1.     

Open reading frame S/L of varicella-zoster virus encodes a cytoplasmic protein expressed in infected cells

We report the discovery of a novel gene in the varicella-zoster virus (VZV) genome, designated open reading frame (ORF) S/L. This gene, located at the left end of the prototype VZV genome isomer, expresses a polyadenylated mRNA containing a splice within the 3' untranslated region in virus-infected cells. Sequence analysis reveals significant differences between the ORF S/Ls of wild-type and attenuated strains of VZV. Antisera raised to a bacterially expressed portion of ORF S/L reacted specifically with a 21-kDa protein synthesized in cells infected with a VZV clinical isolate and with the original vaccine strain of VZV (Oka-ATCC). Cells infected with other VZV strains, including a wild-type strain that has been extensively passaged in tissue culture and commercially produced vaccine strains of Oka, synthesize a family of proteins ranging in size from 21 to 30 kDa that react with the anti-ORF S/L antiserum. MeWO cells infected with recombinant VZV harboring mutations in the C-terminal region of the ORF S/L gene lost adherence to the stratum and adjacent cells, resulting in an altered plaque morphology. Immunohistochemical analysis of VZV-infected cells demonstrated that ORF S/L protein localizes to the cytoplasm. ORF S/L protein was present in skin lesions of individuals with primary or reactivated infection and in the neurons of a dorsal root ganglion during virus reactivation.     

Expression strategy of a phlebovirus: biogenesis of proteins from the Rift Valley fever virus M segment.

The middle (M) RNA segment of Rift Valley fever virus (RVFV) encodes four proteins: the major viral glycoproteins G2 and G1, a 14-kilodalton (kDa) protein, and a 78-kDa protein. These proteins are derived from a single large open reading frame (ORF) present in the virus-complementary M-segment mRNA. We used recombinant vaccinia viruses in which sequences representing the M-segment ORF were engineered as a surrogate system to study phlebovirus protein expression. To investigate the translational initiation codon requirements for synthesis of these proteins, we constructed a series of vaccinia virus recombinants containing specific sequence changes which eliminated select ATG codons found in the region of the ORF preceding the mature glycoprotein-coding sequences (the preglycoprotein region). Examination of phleboviral proteins synthesized in cells infected with these vaccinia virus recombinants clearly showed that the first ATG of the ORF was required for the production of the 78-kDa protein, while synthesis of the 14-kDa protein was absolutely dependent on the second in-phase ATG codon. Efficient biosynthesis of glycoprotein G2 was shown to depend on one or more ATG codons within the preglycoprotein region, but not the first one of the ORF. Synthesis of about one-half of the total glycoprotein G1 was affected by the amino acid changes that eliminated ATG codons, while production of the remainder appeared to be independent of all ATG codons in the preglycoprotein region. These data indicated that the means for glycoprotein G1 biosynthesis was distinct from those of the other three M-segment gene products. The results presented herein suggest that a surprisingly complex expression strategy is employed by the RVFV M segment. Although the full nature of the mechanisms involved in the biogenesis of the four RVFV M-segment proteins remains unclear, it does involve the use of at least two (ATG codons 1 and 2), and likely more, distinct translation start sites within the same ORF to produce its complete complement of gene products.     

Sequence analysis of a gene cluster involved in metabolism of 2,4,5-trichlorophenoxyacetic acid by Burkholderia cepacia AC1100.

Burkholderia cepacia AC1100 utilizes 2,4,5-trichlorophenoxyacetic acid (2,4,5-T) as a sole source of carbon and energy. PT88 is a chromosomal deletion mutant of B. cepacia AC1100 and is unable to grow on 2,4,5-T. The nucleotide sequence of a 5.5-kb chromosomal fragment from B. cepacia AC1100 which complemented PT88 for growth on 2,4,5-T was determined. The sequence revealed the presence of six open reading frames, designated ORF1 to ORF6. Five polypeptides were produced when this DNA region was under control of the T7 promoter in Escherichia coli; however, no polypeptide was produced from the fourth open reading frame, ORF4. Homology searches of protein sequence databases were performed to determine if the proteins involved in 2,4,5-T metabolism were similar to other biodegradative enzymes. In addition, complementation studies were used to determine which genes were essential for the metabolism of 2,4,5-T. The first gene of the cluster, ORF1, encoded a 37-kDa polypeptide which was essential for complementation of PT88 and showed significant homology to putative trans-chlorodienelactone isomerases. The next gene, ORF2, was necessary for complementation and encoded a 47-kDa protein which showed homology to glutathione reductases. ORF3 was not essential for complementation; however, both the 23-kDa protein encoded by ORF3 and the predicted amino acid sequence of ORF4 showed homology to glutathione S-transferases. ORF5, which encoded an 11-kDa polypeptide, was essential for growth on 2,4,5-T, but the amino acid sequence did not show homology to those of any known proteins. The last gene of the cluster, ORF6, was necessary for complementation of PT88, and the 32-kDa protein encoded by this gene showed homology to catechol and chlorocatechol-1,2-dioxygenases.     

The DNA [adenine-N6]methyltransferase (Dam) of bacteriophage T4

A functional bacteriophage T4 dam+ gene, which specifies a DNA [adenine-N6]methyltransferase (Dam), was cloned on a 1.8-kb HindIII fragment [Schlagman and Hattman, Gene 22 (1983) 139-156]. Sequence analysis [Macdonald and Mosig, EMBO J. 3 (1984) 2863-2871] revealed two overlapping in-phase open reading frames (ORFs). The 5' proximal ORF initiates translation at an AUG and encodes a 30-kDa polypeptide, whereas the downstream ORF initiates translation at a GUG and encodes a 26-kDa polypeptide. Analysis of BAL 31 deletions in our original dam+ clone has verified that at least one of these overlapping ORFs, in fact, encodes T4 Dam. To investigate where T4 Dam translation is initiated, we have constructed plasmids in which a tac or lambda PL promoter is placed 5' to either the longer ORF or just the shorter ORF. Only clones which contain a promoter in front of the longer ORF produce active T4 Dam. This indicates that the 26-kDa polypeptide alone cannot be T4 Dam. Additional experiments suggest that only the 30-kDa polypeptide is required for enzyme activity and that the shorter ORF is not translated in plasmid-carrying cells. We also present evidence that T4 Dam is capable of methylating 5'-GATC-3', GATm5C, and GAThmC sequences; non-canonical sites (e.g., GACC) are also methylated, but much less efficiently.     

Transfection of a mutant regulatory subunit gene of cAMP-dependent protein kinase causes increased drug sensitivity and decreased expression of P-glycoprotein

Wild-type Chinese hamster ovary (CHO) cells were transfected with a DNA clone (MT-REV, site A) carrying a mouse gene for a dominant mutant regulatory subunit (RI) gene of cAMP-dependent protein kinase (PKA) from S49 cells along with a marker for G418 resistance. G418-resistant transfectant clone R-2D1 was resistant to 8-Br-cAMP-induced growth inhibition and morphological changes. The cells also did not phosphorylate a 50-kDa protein after cAMP stimulation and had decreased PKA activity, both characteristics of PKA mutants. Northern blot analysis indicated that clone R-2D1 was actively transcribing the MT-REV (site A)-specific RNA. We also tested clone R-2D1 for sensitivity to certain natural product hydrophobic drugs and found increased sensitivity to several drugs including adriamycin. Hypersensitivity to these drugs has previously been shown by us to be a characteristic of a CHO PKA mutant cell line. Expression of the mutant RI gene is also associated with a decrease in expression of the multidrug resistance associated P-glycoprotein (gp170) mRNA and protein. These results show that the PKA mutant RI gene from S49 cells acts as a dominant mutation to reduce the total PKA activity in the CHO transfectants as it does in mouse S49 cells. This study also confirms that reduced PKA activity modulates the basal multidrug resistance of these cells, apparently by causing decreased expression of the mdr gene at the protein and mRNA level.