An additional family of VH sequences in the channel catfish.

A heavy chain variable region cDNA sequence of the channel catfish (Ictalurus punctatus) prototypical for a new VH family (approximately 20 or more members) is presented. The nucleotide and inferred amino acid sequences differ by 32-52% and 41-68%, respectively, from those for the already described catfish VH families.     

Structure and evolution of mammalian VH families

Antibodies are encoded by a limited number of germline gene segments that undergo somatic diversification through rearrangement and mutation. Because these mutation processes are efficient, it is widely believed that there is little environmental selection pressure for the maintenance of specific antibody gene sequences. We have performed pairwise comparisons of known germline (as opposed to somatically generated) antibody VH elements with the hope of identifying conserved structural features common to sets of VH gene segments. These studies reveal that VH families arose prior to the mammalian radiation and have since been conserved, that this conservation appears to reflect selection at the level of protein sequence, and that the conserved regions are discretely localized on a solvent-exposed face of the heavy chain, at some distance from the antibody combining site. A family-specific region was also identified within the recombinase recognition sequences. Our results provide a context for theories that address the physiological significance of variations in VH family utilization during the development of the immune repertoire.     

Organization of the murine immunoglobulin VH complex: placement of two new VH families (VH10 and VH11) and analysis of VH family clustering and interdigitation

During B cell development, there is an ordered expression of heavy chain variable region (VH) genes during ontogeny such that JH proximal VH genes are rearranged and expressed before the more JH distal VH genes. Thus, the relative chromosomal position of VH genes is biologically significant. We have previously employed deletion mapping to order the nine described murine VH gene families as follows: 3609-J558-(J606/VGAM3-8/S107)-3660-(X24/Q52/7183 ). (Families within parentheses were not mapped relative to each other.) In this report we continue this analysis by mapping two recently described heavy chain variable region gene families (VH10 and VH11). VH10 is located at the JH proximal end of the major cluster of J558 VH gene segments. VH11 (a very small family) is intermingled with the 3660 family. Although in general VH genes are thought to be clustered, we and others have reported some interspersion between families. To further address this issue, we have analyzed 80 recombinant phage clones containing J558 VH gene segments for the presence of other VH family genes. Our data indicate that the J558 and 3609 VH families are extensively intermingled as has recently been described for the most JH proximal Q52 and 7183 families.     

Genomic organization, gene duplication, and expression analysis of interleukin-1beta in channel catfish (Ictalurus punctatus)

Interleukin-1beta (IL-1beta) is one of the pivotal early response pro-inflammatory cytokines that enables organisms to respond to infection and induces a cascade of reactions leading to inflammation. In spite of its importance and two decades of studies in the mammalian species, genes encoding IL-1beta were not identified from non-mammalian species until recently. Recent research, particularly with genomic approaches, has led to sequencing of IL-1beta from many species. Clinical studies also suggested IL-1beta as an immunoregulatory molecule potentially useful for enhancing vaccination. However, no IL-1beta genes have been identified from channel catfish, the primary aquaculture species from the United States. In this study, we identified two distinct cDNAs encoding catfish IL-1beta. Their encoding genes were identified, sequenced, and characterized. The catfish IL-1beta genes were assigned to bacterial artificial chromosome (BAC) clones. Genomic studies indicated that the IL-1beta genes were tandemly duplicated on the same chromosome. Phylogenetic analysis of various IL-1beta genes indicated the possibility of recent species-specific gene duplications in channel catfish, and perhaps also in swine and carp. Expression analysis indicated that both IL-1beta genes were expressed, but exhibited distinct expression profiles in various catfish tissues, and after bacterial infection with Edwardsiella ictaluri.     

Detection of new mutations in six out of 10 Swiss HNPCC families by genomic sequencing of the hMSH2 and hMLH1 genes.

The cancer predisposition in most HNPCC families is believed to be associated with mutations in the human mismatch repair gene homologues hMSH2 and hMLH1. We searched for mutations in our collection of 10 Swiss HNPCC families by sequencing the exons and exon/intron boundaries of the hMSH2 and hMLH1 genes. In four families we found different mutations which are expected to lead to protein truncations of either the hMSH2 or the hMLH1 proteins owing to premature in frame stop codons or splice defects. In two more families we detected mutations leading to an amino acid deletion and an amino acid substitution in an evolutionary conserved residues respectively. None of these mutations has been reported in other families, which is consistent with the notion that HNPCC associated hMSH2 and hMLH1 mutations are heterogeneous and there is no striking founder effect in the Swiss population. Whenever this could be investigated, the presence of the mutations was confirmed in other family members who showed manifestations of HNPCC. Interestingly, an obligate carrier in one of the families developed a brain tumour at the age of 29, histologically verified as a glioblastoma multiforme, which was recently linked to HNPCC in the context of Turcot's syndrome.     

Structure/activity relationships in the L-alanine taste receptor system of the channel catfish, Ictalurus punctatus.

In order to understand the molecular determinants of amino acid taste receptor binding and activation, structure/activity studies were performed using analogs of L-alanine in a competitive ligand binding assay and a taste neurophysiological preparation. The presence of both the amine and carboxylic acid in a charged and unhindered form is a primary requisite for a ligand to both bind and activate L-alanine receptors. Although a number of carboxylic acid derivatives are moderately good stimuli, their neural activity derives from action at receptors different from L-alanine receptors. Of the molecular parameters examined, chirality and molecular volume of the side chain are the most important factors in determining the binding and stimulatory efficacy of L-amino acid taste stimuli. Electronegativity of the side chain did not correlate with receptor site binding. Heterologous ligand-induced enhancement of the binding of L-[3H]alanine by a purified taste membrane preparation is described. Neurophysiological experiments support the hypothesis that this phenomenon may be a basis of peripheral sensory interactions.     

High connectivity, natural antibodies preferentially use 7183 and QUPC 52 VH families

Twenty-seven B cell hybridomas derived from spleen cells of nonimmunized, 6-day-old BALB/c mice and previously characterized serologically were screened for VH-gene expression: hybridomas expressing the two most D-proximal VH-gene families, 7183 and QUPC 52 were over-represented in this sample. Strikingly, clones serologically characterized by high degree of degenerate "specificity" and high idiotypic connectivity were found to use almost exclusively these two VH-gene families. As these serological properties are a unique feature of "natural" antibody repertoires, the results may reflect a functional relationship between expression of these VH genes and activation of B cells in the internal environment, particularly in the context of idiotypic interactions.     

Cloning and expression of the AID gene in the channel catfish

A full-length activation-induced cytidine deaminase (AID or Aicda) cDNA has been obtained from the channel catfish (Ictalurus punctatus). A single open reading frame predicts a 209 amino acid protein that has 57% identity and 73% similarity with the AID proteins of mouse and human. All residues that have previously been found to be critical for deamination, as well as for somatic hypermutation, are conserved in the catfish AID. These residues are also conserved in AID proteins predicted, from genome database sequences, to be expressed in Fugu and zebrafish. The catfish AID is expressed at low levels in spleen, kidney, intestine and fin margins, but not in muscle, liver or brain. Immunoglobulin heavy chain (IgH) is also expressed in the tissues where AID is expressed. The 'ectopic' expression of AID in non-lymphoid tissue was unexpected and not readily explained. However, the identification of a fish AID gene will allow us to determine the tissue architecture and locations for affinity maturation in fish.     

Nucleotide sequence, genome organization and phylogenetic analysis of Strawberry pallidosis associated virus, a new member of the genus Crinivirus

Summary. The complete nucleotide sequence of Strawberry pallidosis associated virus (SPaV), a newly identified member of the genus Crinivirus, family Closteroviridae has been determined. RNA 1 is 8067 nucleotides long and encodes at least three open reading frames (ORFs). The first ORF (ORF 1a) specifies a multifunctional protein that has papain-like proteinase, methyltransferase and RNA helicase domains. The RNA-dependent-RNA polymerase is encoded in ORF 1b and is probably expressed by a +1 ribosomal frameshift. The 3 ORF of RNA 1 encodes a small protein with two potential transmembrane helices. RNA 2 is 7979 nucleotides long and encodes 8 ORFs, similar in amino acid sequence and arrangement with those of other criniviruses. SPaV encodes the largest structural protein of closteroviruses sequenced to date as the minor coat protein of the virus has molecular mass of approximately 80kDa. The 3 non-translated regions share nucleotide sequence identities of about 56% and the predicted folding of the non-translated regions is similar. Phylogenetic analyses reveal that SPaV is related most closely to Abutilon yellows virus and Beet pseudo-yellows virus, another virus that has been identified recently to cause identical symptoms on strawberry indicator plants as SPaV.     

MHC RFLP analyses in channel catfish full-sibling families: identification of the role of MHC molecules in spontaneous allogeneic cytotoxic responses

Genes encoding MHC class I and II molecules have been identified in a number of fish species, including the channel catfish, but there is still a dearth of knowledge concerning their functional roles in teleost immune responses. This has in part been due to a lack of appropriate MHC class I and II matched and mismatched animals. To identify such animals, MHC segregation and linkage studies in the channel catfish were undertaken. The results of restriction fragment length polymorphism and fluorescent in situ hybridization studies showed that all the MHC class II genes are linked and most if not all MHC class I genes are linked. These studies also demonstrated that in catfish, as in other teleosts, MHC class I and II genes are not linked. Consequently, catfish matched and mismatched for MHC class I and II genes were identified and preliminary functional studies indicate that spontaneous non-specific allogeneic cytotoxic responses are likely mediated by differences in MHC class I, but not class II, region molecules.     

Agglutination and bactericidal responses of the channel catfish to

Intraperitoneal immunization of channel catfish, , with in Freund's complete adjuvant produced persistent agglutinating antibody titers of hundreds of thousands during a 1-year response. Bactericidal activity of 2 high-titered sera averaged 30% and 20%. Post-immunization bactericidal activity varied little and remained within 10% of pre-immunization bactericidal activity. Fresh channel catfish serum from unimmunized catfish exhibited 100% bactericidal activity against . Lysozyme was present in fresh channel catfish serum at 1.8 μ/ml serum or 34 ng lysozyme/mg protein.     

The human immunoglobulin VH7 gene family consists of a small,polymorphic group of six to eight gene segments dispersed throughout the VH locus

In this report we describe the analysis and mapping of members of the human immunoglobulin V_H7 gene family. V_H7 and V_H1 gene segments are closely related, with individual gene segments sharing between 78% and 82% sequence identity. Divergence from V_H1 gene sequence occurs as an abrupt event at the boundary between framework region (FR) 2 and complementarity-determining region (CDR) 2 and continues through a major portion of FR 3. We used polymerase chain reaction amplification to create a 162-base pair probe spanning the family-specific region of CDR 2 and FR 3 that proved suitable for standard Southern analysis of genomic DNA. The V_H7 gene family was found to be a small but discrete V_H gene family consisting of five to eight germ-line elements, of which at least three are polymorphic. Four different V_H7 gene segments were cloned from the germ line of a single individual, and assigned to specific restriction fragments by sequence-specific hybridization. Two of the four V_H7 elements were pseudogenes. The pattern of sequence variation in these and other known pseudogenes suggests that these nonfunctional elements may play a role in the evolution of novel V_H families. A combination of one and two-dimensional pulsed field gel electrophoresis was employed to map the chromosomal location of all of these V_H7 elements. Individual V_H7 gene segments were found to be dispersed over a region of at least 940 kb of DNA, and interspersed with members from other V_H gene families. The polymorphism of the V_H7 gene segments and their scattered location throughout the V_H locus makes them potentially useful markers for mapping and linkage studies.