Climate Change and Knowledge Communities

19 p.

Organization and function of the phage Lambda chromosome

The process of prophage integration by phage λ and the function and structure of the chromosomal elements required for λ integration have been studied with the use of λ deletion mutants. Since att^φ, the substrate of the integration enzymes, is not essential for λ growth, and since att^φ resides in a portion of the λ chromosome which is not necessary for vegetative growth, viable λ deletion mutants were isolated and examined to dissect the structure of att^φ.

Deletion mutants were selected from wild type populations by treating the phage under conditions where phage are inactivated at a rate dependent on the DNA content of the particles. A number of deletion mutants were obtained in this way, and many of these mutants proved to have defects in integration. These defects were defined by analyzing the properties of Int-promoted recombination in these att mutants.

The types of mutants found and their properties indicated that att^φ has three components: a cross-over point which is bordered on either side by recognition elements whose sequence is specifically required for normal integration. The interactions of the recognition elements in Int-promoted recombination between att mutants was examined and proved to be quite complex. In general, however, it appears that the λ integration system can function with a diverse array of mutant att sites.

The structure of att^φ was examined by comparing the genetic properties of various att mutants with their location in the λ chromosome. To map these mutants, the techniques of heteroduplex DNA formation and electron microscopy were employed. It was found that integration cross-overs occur at only one point in att^φ and that the recognition sequences that direct the integration enzymes to their site of action are quite small, less than 2000 nucleotides each. Furthermore, no base pair homology was detected between att^φ and its bacterial analog, att^B. This result clearly demonstrates that λ integration can occur between chromosomes which have little, if any, homology. In this respect, λ integration is unique as a system of recombination since most forms of generalized recombination require extensive base pair homology.

An additional study on the genetic and physical distances in the left arm of the λ genome was described. Here, a large number of conditional lethal nonsense mutants were isolated and mapped, and a genetic map of the entire left arm, comprising a total of 18 genes, was constructed. Four of these genes were discovered in this study. A series of λdg transducing phages was mapped by heteroduplex electron microscopy and the relationship between physical and genetic distances in the left arm was determined. The results indicate that recombination frequency in the left arm is an accurate reflection of physical distances, and moreover, there do not appear to be any undiscovered genes in this segment of the genome.

Genomic organization and sequence analysis of the vomeronasal receptor V2R genes in mouse genome

Two multigene superfamilies, named V1R and V2R, encoding seven-transmembrane-domain G-protein coupled receptors (GPCRs) have been identified as pheromone receptors in mammals. Three V2R gene families have been described in mouse and rat. Here we screened

Social organization and range use in the Yunnan snub-nosed monkey Rhinopithecus bieti

We studied social organization, behavior, and range use of the Yunnan snub-nosed monkey (Rhinopithecus bieti) at Wuyapiya (99 degrees 12'E, 28 degrees 30'N, the People's Republic of China) over 12 months between May 1992 and June 1994. The Wuyapiya band contained greater than or equal to 175 members and had two levels of social organization. At one level, the monkeys formed multifemale, one-male units (OMUs) similar to those of many other colobines. At another level, 15 to 18 OMUs traveled together in a cohesive band. Unlike the bands of other species of Rhinopithecus, the Wuyapiya band of R. bieti did not show seasonal fission-fusion, although some social behavior, such as male-male aggression, was seasonal. With regard to range use, the Wuyapiya band had a large home range and long daily travel distances compared with other colobines. Minimum range size in 1 year at Wuyapiya is 16.25 km(2), although there is no asymptote for range size as a function of observation time. Range size for the Wuyapiya band is 25.25 km(2) over the 2-year study and appeared to cover 100 km(2) between 1985 and 1994. The primary food of R. bieti at Wuyapiya is lichens, which are ubiquitous in fir frees. The multitiered social organization of R. bieti appears to result from the interaction of food resource characters with the forces of mate competition, with band sizes based on female responses to the spatial and temporal characteristics of lichens and subdivisions within bands based on male competition for mates.

Evolutionary Conservation of Dazl Genomic Organization and its Continuous and Dynamic Distribution Throughout Germline Development in Gynogenetic Gibel Carp

To investigate germline development and germ cell specification, we identified a Dazl homolog (CagDazl) from gynogenetic gibel carp (Carassius auratus gibelio). Its cDNA sequence and BAC clone sequence analyses revealed the genomic organization conservation and conserved synteny of the Dazl family members and their neighborhood genes among vertebrates, especially in fish. Moreover, a polyclonal antibody specific to CagDazl was produced and used to examine its expression and distribution throughout germline development at protein level. Firstly, ovary-specific expression pattern of CagDazl was confirmed in adult tissues by RT-PCR and Western blot. In addition, in situ hybridization and immunofluorescence localization demonstrated its specific expression in germ cells, and both its transcript and protein were localized to germ plasm. Then, co-localization of CagDazl and mitochondrial cloud was found, confirming that CagDazl transcript and its protein are germ plasm component and move via METRO pathway during oogenesis. Furthermore, the CagDazl is abundant and continuous throughout germline development and germ cell specification including primordial germ cell (PGC) formation, oogonium differentiation, oocyte development, and embryogenesis, and the dynamic distribution occurs at different development stages. The data suggest that maternal CagDazl might play an important role in gibel carp PGC formation. Therefore, CagDazl is a useful and specific marker for tracing germ plasm and germ cell development in the gynogenetic gibel carp. In addition, in comparison with previous studies in sexual reproduction species, the continuous and dynamic distribution of CagDazl protein in the germ plasm throughout the life cycle seems to have significant implication in sex evolution of vertebrates. J. Exp. Zool. (Mol. Deu. Euol.) 312B:855-871, 2009. (C) 2009 Wiley-Liss, Inc.

Genomic organization and expression analysis of Toll-like receptor 3 in grass carp (Ctenopharyngodon idella)

Toll-like receptor 3 (TLR3) participates in the innate immune response by recognizing viral pathogens. To investigate grass carp immune system responding to GCRV (grass carp reovirus) infection, the full-length cDNA sequence and genomic organization of grass carp TLR3 (CiTLR3) was identified and characterized. The full-length genome sequence of CiTLR3 is composed of 5668 nucleotides, including five exons and four introns. The full-length of CiTLR3 cDNA is 3681 bp in length and encodes a polypeptide of 904 amino acids with an estimated molecular mass of 102,765 Da and a predicted isoelectric point of 8.35. Analysis of the deduced amino acid sequence indicated that CiTLR3 has four main structural domains, including a signal peptide sequence, 14 LRR (leucine-rich repeat) motifs, a transmembrane region and a TIR (Toll/interleukin-1 receptor) domain. It is most similar to the crucian carp (Carassius auratus) TLR3 amino acid sequence with an identity of 99%. Quantitative RT-PCR analysis showed that CiTLR3 transcripts were significantly up-regulated starting at day 1 and continued through day 7 following GCRV infection (P < 0.05). These data implied that CiTLR3 is involved in antiviral defense, provide molecular and functional information for grass carp TLR3, and implicate their role in mediating immune protection against grass carp viral diseases. (C) 2009 Elsevier Ltd. All rights reserved.

Structure, organization and expression of common carp (Cyprinus carpio L.) NKEF-B gene

Natural killer (NK) cell enhancing factor (NKEF) belongs to the newly defined peroxiredoxin (Prx) family. Its functions are to enhance NK cell cytotoxicity and to protect DNA and proteins from oxidative damage. In this study, a partial cDNA sequence of carp NKEF-B was isolated from thymus cDNA library. Subsequently, the full-length cDNA of carp NKEF-B was obtained by means of 3' and 5' RACE, respectively. The full-length cDNA of carp NKEF-B was 1022 bp, consisting of a 73 bp 5'-terminal untranslated region (UTR), a 355 bp T-terminal UTR, and a 594 bp open reading frame coding for a protein of 197 amino acids. Carp NKEF-B contained two consensus Val-Cys-Pro (VCP) motifs and three consensus cysteine (Cys-51, Cys-70 and Cys-172) residues. Sequence comparison showed that the deduced amino acid sequence of carp NKEF-B had an overall similarity of 74-96% to that of other species homologues. Phylogenetic analysis revealed that carp NKEF-B forms a cluster with other known teleost NKEF-Bs. Then, by PCR we obtained a 5.1 -k long genomic DNA of carp NKEF-B containing six exons and five introns. Realtime RT-PCR results showed that carp NKEF-B gene was predominantly detected in kidney and head kidney under un-infected conditions. Whereas under SVCV-infection condition, the expression of NKEF-B gene was significantly increased in blood cells, gill, intestine and spleen, but maintained in liver, and decreased significantly in kidney and head kidney. Finally, the rNKEF-B was constructed and expressed in Escherichia coli. By using an antibody against carp rNKEF-B, immunohistochemical study further indicated that NKEF-B positive cells are mainly some RBCs and a few epithelial cells in gill and intestine, and that under SVCV-infection condition, these positive cells or positive products in their cytoplasm were mainly increased in gill and spleen sections of carp. The results obtained in the present study will help to understand the function of NKEF-B in teleost innate immunity. (C) 2008 Elsevier Ltd. All rights reserved.