Molecular and structural characterization of the domain 2 of hepatitis C virus non-structural protein 5A

Hepatitis C virus (HCV) non-structural protein 5A protein (NS5A), which consists of three functional domains, is involved in regulating viral replication, interferon resistance, and apoptosis. Recently, the three-dimensional structure of the domain 1 was determined. However, currently the molecular basis for the domains 2 and 3 of HCV NS5A is yet to be defined. Toward this end, we expressed, purified the domain 2 of the NS5A (NS5A-D2), and then performed biochemical and structural studies. The purified domain 2 was active and was able to bind NS5B and PKR, biological partners of NS5A. The results from gel filtration, CD analysis, 1D 1H NMR and 2D 1H-15N heteronuclear single quantum correlation (HSQC) spectroscopy indicate that the domain 2 of NS5A appears to be flexible and disordered.     

Mathematical aspects of protein structure determination with NMR orientational restraints

The field of structural biology is becoming increasingly important as new technological developments facilitate the collection of data on the atomic structures of proteins and nucleic acids. The solid-state NMR method is a relatively new biophysical technique that holds particular promise for determining the structures of peptides and proteins that are located within the cell membrane. This method provides information on the orientation of the peptide planes relative to an external magnetic field. In this article, we discuss some of the mathematical methods and tools that are useful in deriving the atomic structure from these orientational data. We first discuss how the data are viewed as tensors, and how these tensors can be used to construct an initial atomic model, assuming ideal stereochemistry. We then discuss methods for refining the models using global optimization, with stereochemistry constraints treated as penalty functions. These two processes, initial model building followed by refinement, are the two crucial steps between data collection and the final atomic model.     

Murine p53 inhibits the function but not the formation of SV40 T antigen hexamers and stimulates T antigen RNA helicase activity

We have characterized the effects of p53 on several biochemical activities of simian virus 40 (SV40) large tumor (T) antigen. While p53 induced a strong inhibition of the T antigen DNA helicase activity, surprisingly, its RNA helicase activity was stimulated. This supports the liklihood that the DNA and RNA helicase activities of T antigen reflect discrete functions. p53 did not significantly affect the ATP-dependent conversion of T antigen monomers to hexamers. However, the ability of these hexamers to assemble on a DNA fragment containing the viral origin was impaired by p53. Thus, these results suggest that p53 inhibits the function but not the formation of T antigen multimers. This conclusion was further supported by the observation that the addition of a purified p53:T antigen complex was as inhihitory as free p53 to the DNA helicase activity of free T antigen. Thus our data indicates that the targets of p53 inhibition are the functional units of T antigen, namely the hexamers.     

Cloning of the human monocarboxylate transporter MCT3 gene: localization to chromosome 22q12.3-q13.2.

Lactate transport across cell membranes is mediated by a family of proton-coupled monocarboxylate transporters (MCTs). The retinal pigment epithelium (RPE) expresses a unique member of this family, MCT3. A portion of the human MCT3 gene was cloned by polymerase chain reaction using primers designed from rat RPE MCT3 cDNA sequence. The human genomic sequence was used to design primers to clone human MCT3 cDNA and to identify a bacterial artificial chromosome clone containing the human MCT3 gene. The human MCT3 cDNA contained a 1512-nucleotide open reading frame with a deduced amino sequence 85% identical to rat MCT3. Comparison of the cDNA and genomic sequences revealed that the MCT3 gene was composed of five exons distributed over 5 kb of DNA. The exon-intron borders were conserved between the human and the chicken MCT3 genes. Using radiation hybrid mapping, the MCT3 gene was mapped to chromosome 22 between markers WI11639 and SGC30687. A search of chromosome 22 in the Sanger Centre database confirmed the location of the human MCT3 gene at 22q12.3-q13.2.     

Ribosomal DNA Intergenic Spacer of the Swimming Crab, Charybdis japonica

We have determined the full sequence of the ribosomal DNA intergenic spacer (IGS) of the swimming crab, Charybdis japonica, by long PCR for the first time in crustacean decapods. The IGS is 5376 bp long and contains two nonrepetitive regions separated by one long repetitive region, which is composed mainly of four subrepeats (subrepeats I, II, III, and IV). Subrepeat I contains nine copies of a 60-bp repeat unit, in which two similar repeat types (60 bp-a and 60 bp-b) occur alternatively. Subrepeat II consists of nine successive repeat units with a consensus sequence length of 142 bp. Subrepeat III consists of seven copies of another 60-bp repeat unit (60 bp-c) whose sequence is complementary to that of subrepeat I. Immediately downstream of subrepeat III is subrepeat IV, consisting of three copies of a 391-bp repeat unit. Based on comparative analysis among the subrepeats and repeat units, a possible evolutionary process responsible for the formation of the repetitive region is inferred, which involves the duplication of a 60-bp subrepeat unit (60 bp-c) as a prototype. Received: 13 April 1999 / Accepted: 2 August 1999     

Ala99ser mutation in RIα Regulatory Subunit of protein kinase A causes reduced kinase activation by cAMP and arrest of hormone-dependent breast cancer cell growth

Expression of the RI regulatory subunit of protein kinase A type I is increased in human cancer cell lines, in primary tumors, in cells after transformation, and in cells upon stimulation of growth. Ala99 (the pseudophosphorylation site) of human RI was replaced with Ser (RI-p) for the structure-function analysis of RI. MCF-7 hormone- dependent breast cancer cells were transfected with an expression vector for the wild-type RI or mutant RI-p. Overexpression of RI-P resulted in suppression of protein kinase A type II, the isozyme of type I kinase, production of kinase exhibiting reduced cAMP activation, and inhibition of cell growth showing an increase in G0/G1 phase of the cell cycle and apoptosis. The wild-type RI overexpression had no effect on protein kinase A isozyme distribution or cell growth. Overexpression of protein kinase A type II regulatory subunit, RII, suppressed RI and protein kinase A type I and inhibited cell growth. These results show that the growth of hormone-dependent breast cancer cells is dependent on the functional protein kinase A type I.     

An ordered EST catalogue and gene expression profiles of cassava (<Emphasis Type="Italic">Manihot esculenta</Emphasis>) at key growth stages

A cDNA library was constructed from the root tissues of cassava variety Huanan 124 at the root bulking stage. A total of 9,600 cDNA clones from the library were sequenced with single-pass from the 5′-terminus to establish a catalogue of expressed sequence tags (ESTs). Assembly of the resulting EST sequences resulted in 2,878 putative unigenes. Blastn analysis showed that 62.6% of the unigenes matched with known cassava ESTs and the rest had no ‘hits’ against the cassava database in the integrative PlantGDB database. Blastx analysis showed that 1,715 (59.59%) of the unigenes matched with one or more GenBank protein entries and 1,163 (40.41%) had no ‘hits’. A cDNA microarray with 2,878 unigenes was developed and used to analyze gene expression profiling of Huanan 124 at key growth stages including seedling, formation of root system, root bulking, and starch maturity. Array data analysis revealed that (1) the higher ratio of up-regulated ribosome-related genes was accompanied by a high ratio of up-regulated ubiquitin, proteasome-related and protease genes in cassava roots; (2) starch formation and degradation simultaneously occur at the early stages of root development but starch degradation is declined partially due to decrease in UDP-glucose dehydrogenase activity with root maturity; (3) starch may also be synthesized in situ in roots; (4) starch synthesis, translocation, and accumulation are also associated probably with signaling pathways that parallel Wnt, LAM, TCS and ErbB signaling pathways in animals; (5) constitutive expression of stress-responsive genes may be due to the adaptation of cassava to harsh environments during long-term evolution.     

HLA association with nasopharyngeal carcinoma in southern Tunisia

In order to study the association of HLA-A, -B and/or DRB1, DQB1 and the nasopharyngeal carcinoma (NPC), 141 patients affected with NPC were typed for the HLA class I by serology method of microlymphocytotoxicity. Among these patients 101 were genotyped for HLA class II system by the PCR-SSP technique. HLA typing results were compared to those of 116 controls. We found that the HLA-A31 and -A33 antigens were significantly more expressed in patients than in the controls (P = 0.016 and 0.010, respectively) and the HLA-A19 antigen, was significantly more frequent in patients when compared to the controls (P = 0.007). The HLA-DRB1*03 and DRB1*13 alleles were significantly more frequent in patients as compared to the controls. The DRB1*01 allele was expressed with a frequency of 20.69% in the controls whereas it was only detected in 3.96% of the NPC patients. Furthermore, the DQB1*05 allele was expressed at a frequency which was significantly less important in affected patient (P = 0.03), whereas, the DQB1*02 allele was more frequent in patients (P = 0.643 × 10⁻⁴). Thus our study revealed a significant increase of HLA-A31, A33, A19, B16, B53 and DRB1*03, DRB1*13 and DQB1*02 alleles in our patients. These markers could play a predisposing role in the development of NPC. In contrast, a decrease of HLA-B14, -B35 and DRB1*01 and DQB1*05 alleles was found suggesting a likely protective effect.