The United States Culture Collection Network (USCCN): Enhancing Microbial Genomics Research through Living Microbe Culture Collections

The mission of the United States Culture Collection Network (USCCN; http://usccn.org) is “to facilitate the safe and responsible utilization of microbial resources for research, education, industry, medicine, and agriculture for the betterment of human kind.” Microbial culture collections are a key component of life science research, biotechnology, and emerging global biobased economies. Representatives and users of several microbial culture collections from the United States and Europe gathered at the University of California, Davis, to discuss how collections of microorganisms can better serve users and stakeholders and to showcase existing resources available in public culture collections.     

Human and murine cytotoxic T cells specific to respiratory syncytial virus recognize the viral nucleoprotein (N), but not the major glycoprotein (G), expressed by vaccinia virus recombinants

The viral antigens recognized by cytotoxic T cells (CTL) have not been defined in most viruses infecting mouse or man. Natural or artificial virus recombinants can be used to determine the antigen specificity of CTL directed against viruses with segmented genomes, such as influenza, but this technique is more difficult to apply to the study of unsegmented viruses. We describe here the use of recombinant vaccinia viruses, containing cDNA corresponding to either the nucleoprotein (N) gene or the major surface glycoprotein (G) gene of human respiratory syncytial virus (RSV), to examine the antigen specificity of anti-RSV cytotoxic T cells from humans and mice. The results demonstrate that the RSV N protein is one of the target antigens for CTL in man and mouse, whereas the G protein was not recognized and can at best represent a minor target antigen for CTL.     

Angiogenesis in Balb/c mice under beta-carotene supplementation in diet

Angiogenesis is a process of new blood vessel formation from pre-existing ones. The most important steps in angiogenesis include detachment, proliferation, migration, homing and differentiation of vascular wall cells, which are mainly endothelial cells and their progenitors. The study focused on the effect of beta-carotene (BC) supplementation (12,000 mg/kg) in the diet on angiogenesis in Balb/c mice. Female Balb/c mice were fed for 5 weeks with two different diets: with BC or without BC supplementation. After 4 weeks of feeding, Balb/c mice were injected subcutaneously with two matrigel plugs with or without basic fibroblast growth factor (bFGF). Six days later, the animals were killed, and the matrigel plugs were used for immunohistochemical staining with CD31 antibody and for gene expression analysis. Microarray and Real-Time PCR data showed down-regulation of genes involved in proliferation and up-regulation of genes encoding inhibitors of apoptosis, proteins regulating cell adhesion, matrix-degrading enzymes and proteins involved in the VEGF pathway. The results of this study demonstrated that BC proangiogenic activity (with or without bFGF) in vivo seemed to be more significantly associated with cells’ protection from apoptosis and their stimulation of chemotaxis/homing than cell proliferation.     

The envelope-associated 22K protein of human respiratory syncytial virus: nucleotide sequence of the mRNA and a related polytranscript

We recently determined that respiratory syncytial virus (strain A2) encodes a fourth unique envelope-associated virion protein that has molecular weight of approximately 24,000, as estimated by gel electrophoresis. The nucleotide sequence of the mRNA encoding this novel protein has now been determined from five cDNA clones, including three that contain the complete mRNA sequence. The complete mRNA sequence is 957 nucleotides, exclusive of polyadenylate, and contains two partially overlapping open reading frames. The 5'-proximal open reading frame is favored for utilization by the criteria of the location and sequence of its translational start site. Furthermore, the calculated molecular weight of the encoded protein, 22,153, is in agreement with the previous estimate of 24,000 for the authentic protein identified by hybrid selection and in vitro translation. The sequence of the predicted protein, now designated the 22K protein, contains 194 amino acids, is relatively hydrophilic, and appears to be the most basic of the respiratory syncytial virus proteins. The mRNA also contains a second, internal open reading frame which would encode a protein of 90 amino acids. However, no evidence for this translation product is known. The first nine nucleotides in the mRNA sequence, 5'-GGGGCAAAU, are identical to the conserved sequence identified previously at the 5' termini of seven other respiratory syncytial viral mRNAs; the sequence at the 3' end of the 22K mRNA, 5'. . . AGUUAUUU-polyadenylate, contains the elements of the previously identified 3'-terminal consensus sequence for respiratory syncytial virus mRNAs, AGUUAA(N)1-4-polyadenylate (P. L. Collins, Y. T. Huang, and G. W. Wertz, Proc. Natl. Acad. Sci. U.S.A. 81:7683-7687). In addition, we present and describe the intergenic sequence of a dicistronic RNA derived from readthrough of the F and 22K protein genes.     

Molecular characterization of the klebicin B plasmid of Klebsiella pneumoniae.

The nucleotide sequence of a bacteriocin-encoding plasmid isolated from Klebsiella pneumoniae (pKlebB-K17/80) has been determined. The encoded klebicin B protein is similar in sequence to the DNase pyocins and colicins, suggesting that klebicin B functions as a nonspecific endonuclease. The klebicin gene cluster, as well as the plasmid backbone, is a chimera, with regions similar to those of pore-former colicins, nuclease pyocins and colicins as well as noncolicinogenic plasmids. Similarities between pKlebB plasmid maintenance functions and those of the colicin E1 plasmid suggest that pKlebB is a member of the ColE1 plasmid replication family.     

Genetic diversity of the attachment protein of subgroup B respiratory syncytial viruses.

Respiratory syncytial (RS) virus causes repeated infections throughout life. Between the two main antigenic subgroups of RS virus, there is antigenic variation in the attachment protein G. The antigenic differences between the subgroups appear to play a role in allowing repeated infections to occur. Antigenic differences also occur within subgroups; however, neither the extent of these differences nor their contributions to repeat infections are known. We report a molecular analysis of the extent of diversity within the subgroup B RS virus attachment protein genes of viruses isolated from children over a 30-year period. Amino acid sequence differences as high as 12% were observed in the ectodomains of the G proteins among the isolates, whereas the cytoplasmic and transmembrane domains were highly conserved. The changes in the G-protein ectodomain were localized to two areas on either side of a highly conserved region surrounding four cysteine residues. Strikingly, single-amino-acid coding changes generated by substitution mutations were not the only means by which change occurred. Changes also occurred by (i) substitutions that changed the available termination codons, resulting in proteins of various lengths, and (ii) a mutation introduced by a single nucleotide deletion and subsequent nucleotide insertion, which caused a shift in the open reading frame of the protein in comparison to the other G genes analyzed. Fifty-one percent of the G-gene nucleotide changes observed among the isolates resulted in amino acid coding changes in the G protein, indicating a selective pressure for change. Maximum-parsimony analysis demonstrated that distinct evolutionary lineages existed. These data show that sequence diversity exists among the G proteins within the subgroup B RS viruses, and this diversity may be important in the immunobiology of the RS viruses.     

Medical geneticists confront ethical dilemmas: cross-cultural comparisons among 18 nations.

To provide a basis for international discussion of ethical problems, we studied responses of medical geneticists in 18 countries to questionnaires about 14 clinical cases and five screening situations. Of 1,053 asked to participate, 677 (64%) responded. There was greater than or equal to 75% consensus on five cases involving (1) disclosure of (1) conflicting diagnostic findings, (2) disclosure of ambiguous results, (3) disclosure of controversial interpretations, (4) protection of mother's confidentiality in cases of false paternity, and (5) nondirective counseling about 45,X and XYY syndrome. A majority (51%-60%) would disclose the diagnosis to relatives at risk for Huntington disease or hemophilia A, against the patient's wishes; would disclose which parent carries a translocation causing Down syndrome; and would disclose XY genotype in a female. As reproductive options for patients with disorders not diagnosable prenatally, 84% would discuss artificial insemination by a donor, 66% would discuss in vitro fertilization with donor egg, and 46% would discuss surrogate motherhood. In all, 85% would perform prenatal diagnosis for (or would refer) parents who refuse abortion, 75% for maternal anxiety, and 42% for selection of fetal sex. Screening questions showed that 72% believed that workplace screening should be voluntary and that results should be confidential.