The S2 gene of equine infectious anemia virus is dispensable for viral replication in vitro

Equine infectious anemia virus (EIAV) contains the simplest genome among lentiviruses in that it encodes only three putative regulatory genes (S1, S2, S3) in addition to the canonical gag, pol, and env genes, presumably reflecting its limited tropism to cells of monocyte/macrophage lineage. Tat and Rev functions have been assigned to S1 and S3, respectively, but the specific function for the S2 gene has yet to be determined. Thus, the function of S2 in virus replication in vitro was investigated by using an infectious molecular viral clone, EIAVUK. Various EIAVUK mutants lacking S2 were constructed, and their replication kinetics were examined in several equine cell culture systems, including the natural in vivo target equine macrophage cells. The EIAV S2 mutants showed replication kinetics similar to those of the parental virus in all of the tested primary and transformed equine cell cultures, without any detectable reversion of mutant genomes. The EIAVUK mutants also showed replication kinetics similar to those of the parental virus in an equine blood monocyte differentiation-maturation system. These results demonstrate for the first time that the EIAV S2 gene is not essential and does not appear to affect virus infection and replication properties in target cells in vitro.     

A low-temperature-responsive gene from barley encodes a protein with single-stranded nucleic acid-binding activity which is phosphorylated in vitro

A low-temperature-responsive gene, blt 801, isolated from a winter barley (Hordeum vulgare L.) cDNA library prepared from leaf meristematic tissue, was sequenced. The deduced amino acid sequence predicts a glycine-rich RNA-binding protein (GR-RNP) which was homology to stress-responsive GR-RNPs from several other plant species. BLT 801 is a two-domain protein, the amino-terminal domain comprises a consensus RNA-binding domain similar to that found in many eukaryotic genes and the carboxy-terminal domain is extremely glycine-rich (68.5% glycine). Blt 801 mRNA also accumulates in response to the phytohormone abscisic acid. The protein encoded by blt 801 has been produced as a recombinant fusion protein using a bacterial expression vector. The fusion protein, a chimaera of glutathione S-transferase and BLT 801, has been used in studies to determine nucleic acid binding and other characteristics. Binding studies with single-stranded nucleic acids show that BLT 801 has affinity for homoribopolymers G, A and U but not C, it also binds to single-stranded DNA and selects RNA molecules containing open loop structures enriched in adenine but low in cytosine. Blt 801 has a consensus motif for phosphorylation by cAMP protein kinase (PKA) at the junction between the two domains which can be phosphorylated by PKA in vitro and which, by analogy to animal studies, may have significance for controlling enzyme function.     

The gene encoding the elongation factor P protein is essential for viability and is required for protein synthesis

Elongation factor P (EFP) is a protein that stimulates the peptidyltransferase activity of fully assembled 70 S prokaryotic ribosomes and enhances the synthesis of certain dipeptides initiated by N-formylmethionine. This reaction appears conserved throughout species and is promoted in eukaryotic cells by a homologous protein, eIF5A. Here we ask whether the Escherichia coli gene encoding EFP is essential for cell viability. A kanamycin resistance (KanR) gene was inserted near the N-terminal end of the efp gene and was cloned into a plasmid, pMAK705, that has a temperature-sensitive origin of replication. After transformation into a recA+ E. coli strain, temperature-sensitive mutants were isolated, and their chromosomal DNA was sequenced. Mutants containing the efp-KanR gene in the chromosome grew at 33 degrees C only in the presence of the wild-type copy of the efp gene in the pMAK705 plasmid and were unable to grow at 44 degrees C. Incorporation of various isotopes in vivo suggests that translation is impaired in the efp mutant at 44 degrees C. At 44 degrees C, mutant cells are severely defective in peptide-bond formation. We conclude that the efp gene is essential for cell viability and is required for protein synthesis.     

The tip of the hydrophobic hairpin of colicin U is dispensable for colicin U activity but is important for interaction with the immunity protein

The hydrophobic C terminus of pore-forming colicins associates with and inserts into the cytoplasmic membrane and is the target of the respective immunity protein. The hydrophobic region of colicin U of Shigella boydii was mutated to identify determinants responsible for recognition of colicin U by the colicin U immunity protein. Deletion of the tip of the hydrophobic hairpin of colicin U resulted in a fully active colicin that was no longer inactivated by the colicin U immunity protein. Replacement of eight amino acids at the tip of the colicin U hairpin by the corresponding amino acids of the related colicin B resulted in colicin U(575-582ColB), which was inactivated by the colicin U immunity protein to 10% of the level of inactivation of the wild-type colicin U. The colicin B immunity protein inactivated colicin U(575-582ColB) to the same degree. These results indicate that the tip of the hydrophobic hairpin of colicin U and of colicin B mainly determines the interaction with the corresponding immunity proteins and is not required for colicin activity. Comparison of these results with published data suggests that interhelical loops and not membrane helices of pore-forming colicins mainly interact with the cognate immunity proteins and that the loops are located in different regions of the A-type and E1-type colicins. The colicin U immunity protein forms four transmembrane segments in the cytoplasmic membrane, and the N and C termini face the cytoplasm.     

MMS2, encoding a ubiquitin-conjugating-enzyme-like protein, is a member of the yeast error-free postreplication repair pathway

Among the three Saccharomyces cerevisiae DNA repair epistasis groups, the RAD6 group is the most complicated and least characterized, primarily because it consists of two separate repair pathways: an error-free postreplication repair pathway, and a mutagenesis pathway. The rad6 and rad18 mutants are defective in both pathways, and the rev3 mutant affects only the mutagenesis pathway, but a yeast gene that is involved only in error-free postreplication repair has not been reported. We cloned the MMS2 gene from a yeast genomic library by functional complementation of the mms2-1 mutant [Prakash, L. & Prakash, S. (1977) Genetics 86, 33-55]. MMS2 encodes a 137-amino acid, 15.2-kDa protein with significant sequence homology to a conserved family of ubiquitin-conjugating (Ubc) proteins. However, Mms2 does not appear to possess Ubc activity. Genetic analyses indicate that the mms2 mutation is hypostatic to rad6 and rad18 but is synergistic with the rev3 mutation, and the mms2 mutant is proficient in UV-induced mutagenesis. These phenotypes are reminiscent of a pol30-46 mutant known to be impaired in postreplication repair. The mms2 mutant also displayed a REV3-dependent mutator phenotype, strongly suggesting that the MMS2 gene functions in the error-free postreplication repair pathway, parallel to the REV3 mutagenesis pathway. Furthermore, with respect to UV sensitivity, mms2 was found to be hypostatic to the rad6Delta1-9 mutation, which results in the absence of the first nine amino acids of Rad6. On the basis of these collective results, we propose that the mms2 null mutation and two other allele-specific mutations, rad6Delta1-9 and pol30-46, define the error-free mode of DNA postreplication repair, and that these mutations may enhance both spontaneous and DNA damage-induced mutagenesis.     

Structural organization of a gene encoding a novel calmodulin-binding kinesin-like protein from Arabidopsis

Kinesin-like calmodulin-binding protein (KCBP) is a recently identified microtubule motor protein that appears to be unique to plants. Here we report isolation and sequence analysis of a gene encoding Arabidopsis KCBP. KCBP gene contains 21 exons and 20 introns. All exons except exon 3 are short (94-272 nt). Exons 1-9 code for the globular tail region whereas the coiled-coil region is coded by exons 10-15. The conserved motor domain is coded by exons 16-20. Calmodulin-binding domain that is present in the C-terminal region of the protein and unique to KCBP is coded by the last exon. The size of introns ranged from 71 (intron 17) to 320 (intron 19) nucleotides. As in most plant introns, the content of AT is very high in all introns (up to 76%). Phylogenetic analysis of KCBP using motor domain sequence grouped KCBP with other known C-terminal microtubule motor proteins. However, Arabidopsis KCBP together with its homologs from potato and tobacco constitute a distinct group within the C-terminal subfamily of motors which is consistent with structural and functional features of KCBP.     

The yhhP gene encoding a small ubiquitous protein is fundamental for normal cell growth of Escherichia coli

H-NS is a major constituent of the Escherichia coli nucleoid, whereas sigmaS is a stress-induced sigma factor. An hns null mutation affects the cellular content of sigmaS in such a way that a remarkable accumulation of sigmaS is observed in the logarithmic growth phase, which results in enhanced expression of a number of sigmaS-dependent genes, including the katE gene. We isolated an extragenic mutation that affects the expression of the katE-lacZ fusion gene in the deltahns background. The relevant gene was identified as yhhP, which encodes a small polypeptide of 81 amino acids. Lesion of this gene seemed to affect the stability of sigmaS. A deletion analysis of yhhP revealed that this small protein plays a fundamental role in the general physiology of E. coli. The yhhP-deficient cell is not capable of growing in standard laboratory rich medium (i.e., Luria broth), resulting in the formation of filamentous cells. Homologs of this intriguing protein occur in a wide variety of bacterial species, including archaeal species.     

hgpB, a gene encoding a second Haemophilus influenzae hemoglobin- and hemoglobin-haptoglobin-binding protein

Haemophilus influenzae requires heme for growth and can utilize both hemoglobin and hemoglobin-haptoglobin as heme sources. We previously identified a hemoglobin- and hemoglobin-haptoglobin-binding protein, HgpA, in H. influenzae HI689. Mutation of hgpA did not affect binding or utilization of either heme source. The hgpA mutant exhibited loss of a 120-kDa protein and increased expression of a 115-kDa protein. These data suggested that at least one other gene product is involved in binding of these heme sources by H. influenzae. A 3.2-kbp PCR product derived from HI689 was cloned. The nucleotide sequence indicated a separate, distinct gene with high homology to hgpA, which would encode a 115-kDa protein. Primers were designed for directional cloning of the structural gene in the correct reading frame. Sonicates of induced Escherichia coli harboring the cloned open reading frame bound both hemoglobin and hemoglobin-haptoglobin. An insertion/deletion mutant of H. influenzae at the newly identified locus, designated hgpB, was constructed. The 115-kDa protein was not detected in the mutant after affinity purification using biotinylated hemoglobin. An hgpA hgpB double-mutant strain exhibited a reduced ability to utilize hemoglobin-haptoglobin, although it was unaltered in the ability to utilize hemoglobin. Affinity isolation of hemoglobin-binding proteins from the double mutant resulted in isolation of an approximately 120-kDa protein. Internal peptide sequencing revealed this protein to be a third distinct protein, highly homologous to HgpA and HgpB. In summary a second hemoglobin- and hemoglobin-haptoglobin-binding protein of H. influenzae has been identified and characterized, and the presence of an additional protein of similar function has been revealed.     

An ascidian gene encoding an SH2-domain protein is expressed in the notochord cells of the embryo

OBJECTIVE: The purpose of this study is to explore the apparent social mediation of a postpartum somatic illness, na tadoka ni vasucu, occurring among ethnic Fijian women. METHOD: During their first two postpartum days, 85 consecutive newly delivered ethnic Fijian women were recruited for a prospective study on na tadoka ni vasucu at the Sigatoka District Hospital in Nadroga, Fiji. Subjects underwent translated structured interviews and responded to the Kellner Symptom Questionnaire and to visual analog scales to assess social supports and occurrence of mood symptoms or an episode of na tadoka ni vasucu in the postpartum period. Semistructured ethnographic interviews were also conducted with subjects who reported an episode of na tadoka ni vasucu. Data were collected in the initial postpartum days and again at 2 to 5 months postpartum; 82 women completed the study. RESULTS: Na tadoka ni vasucu is a somatic syndrome occurring in 9% (N = 7) of this sample. Both quantitative and narrative data demonstrate that this syndrome is associated with perceived inferior social supports. Despite its relatively infrequent occurrence and benign clinical course, the disorder is a subject of serious social concern within the Fijian community. CONCLUSIONS: Although na tadoka ni vasucu seems to be clinically trivial, because of its cultural salience it is nonetheless able to mobilize intensive social surveillance and care for the postpartum mother. The moral concern generated by this culturally marked disorder, as well as its association with perceived inferior social supports, suggest a dialectical relationship between somatic idiom and its social context.     

Cloning of aas, a gene encoding a Staphylococcus saprophyticus surface protein with adhesive and autolytic properties

A gene encoding a novel cell wall-associated protein of Staphylococcus saprophyticus that binds fibronectin and to sheep erythrocytes has been cloned and sequenced. The 4392 bp open reading frame codes for an amino acid sequence that is quite similar to the Atl, an autolysin, of Staphylococcus aureus and to the AtlE of S. epidermidis. The two regions of most pronounced homology code for an N-acetyl-muramyl-L-alanine amidase and for an endo-beta-N-acetyl-D-glucosaminidase. The cloned protein lysed cells of S. saprophyticus and Micrococcus luteus exogenously. Subcloning localized the enzymatic activities to the regions of high homology and demonstrated that the interposed sequence is responsible for the adhesive activities. Two allelic replacement mutants were constructed that lacked autolytic activity and adhesive properties. The N-terminal portion of the protein contains seven highly conserved, contiguous repeats with no similarity to published sequences. It lacks the motifs typical of Gram-positive surface proteins and shows a different overall organization. This autolysin/adhesin of S. saprophyticus (Aas) appears to represent a new class of staphylococcal adhesins.     

DHR3, an ecdysone-inducible early-late gene encoding a Drosophila nuclear receptor, is required for embryogenesis

Response to the steroid hormone ecdysone in Drosophila is controlled by genetic regulatory hierarchies that include eight members of the nuclear receptor protein family. The DHR3 gene, located within the 46F early-late ecdysone-inducible chromosome puff, encodes an orphan nuclear receptor that recently has been shown to exert both positive and negative regulatory effects in the ecdysone-induced genetic hierarchies at metamorphosis. We used a reverse genetics approach to identify 11 DHR3 mutants from a pool of lethal mutations in the 46F region on the second chromosome. Two DHR3 mutations result in amino acid substitutions within the conserved DNA binding domain. Analysis of DHR3 mutants reveals that DHR3 function is required to complete embryogenesis. All DHR3 alleles examined result in nervous system defects in the embryo.     

Infection-derived Enterococcus faecalis strains are enriched in esp, a gene encoding a novel surface protein

In the present study, the levels of soluble adhesion molecules P- and E-selectin, intercellular adhesion molecule-1 (ICAM-1), vascular adhesion molecule-1 (VCAM-1) and of other markers of endothelial activation or injury, such as thrombomodulin, von Willebrand factor (vWF), as well as homocysteine, were prospectively investigated in 71 patients (21 women, 50 men, age 68+/-13) with predominantly femoropopliteal peripheral arterial occlusive disease (PAOD, stage II-IV, Fontaine) before and after percutaneous transluminal angioplasty (PTA). Thirty patients (42.3%) developed restenosis within 6 months, defined as a > 50% reduction of the lumen diameter at the site of PTA. At entry in the study, 46% and 58% of all patients had higher than normal levels of soluble P-selectin and VCAM-1, respectively. Thrombomodulin (P < 0.01) measured at entry, was significantly higher in patients who developed late restenosis, with trends for higher values for P-selectin, VCAM-1 and vWF. The relative risks for developing restenosis were 2.41 (CI95%: 1.23-4.75) and 1.54 (CI95%: 0.98-2.72) for thrombomodulin and P-selectin, respectively. Soluble P-selectin and the severity of PAOD (Fontaine stage III/IV) were found to be statistically indicative factors for late restenosis in a logistic regression risk factor analysis with an overall predictive value of 72%. At 6 months, those who developed restenosis had also higher soluble P-selectin (P < 0.01), VCAM-1 (P < 0.05) and a trend for higher thrombomodulin. Homocysteine was elevated in 52% of the patients at entry but neither was it associated with higher restenosis rates nor did it correlate with the levels of thrombomodulin or the other adhesion molecules. These findings indicate that patients with PAOD have to a significant proportion, elevated levels of circulating soluble adhesion molecules and markers of endothelial activation occurring in concert with an ongoing atherosclerotic process.     

Amplification and over-expression of OZF, a gene encoding a zinc finger protein, in human pancreatic carcinomas

The OZF gene encodes a protein consisting of 10 zinc finger motifs and is located on chromosome 19q3.1. We report here the amplification and over-expression of the OZF gene in pancreatic carcinomas. Increased gene copy number was detected in 3 of 12 tumour cell lines and 2 of 12 primary pancreatic carcinomas. Expression was detected in all cell lines, and the gene was over-expressed in cell lines with OZF gene amplification. Five of 8 tumours, including 2 primary tumours with OZF gene amplification, displayed high levels of OZF protein, whereas normal pancreas expressed low levels. Immuno-histochemical analysis showed that expression was restricted to tumour cells. Thus, high-level expression of OZF is frequent in pancreatic carcinomas and may contribute to the development or progression of this tumour.