Characterization of axolotl lampbrush chromosomes by fluorescence in situ hybridization and immunostaining.

The lampbrush chromosomes (LBCs) in oocytes of the Mexican axolotl (Ambystoma mexicanum) were identified some time ago by their relative lengths and predicted centromeres, but they have never been associated completely with the mitotic karyotype, linkage maps or genome assembly. We identified 9 of the axolotl LBCs using RNAseq to identify actively transcribed genes and 13 BAC (bacterial artificial clone) probes containing pieces of active genes. Using read coverage analysis to find candidate centromere sequences, we developed a centromere probe that localizes to all 14 centromeres. Measurements of relative LBC arm lengths and polymerase III localization patterns enabled us to identify all LBCs. This study presents a relatively simple and reliable way to identify each axolotl LBC cytologically and to anchor chromosome-length sequences (from the axolotl genome assembly) to the physical LBCs by immunostaining and fluorescence in situ hybridization. Our data will facilitate a more detailed transcription analysis of individual LBC loops.

A MEQ-deleted Marek's disease virus cloned as a bacterial artificial chromosome is a highly efficacious vaccine.

The Marek's disease virus (MDV) induces T-cell tumors in susceptible chickens. Of the 80 to 100 known MDV genes, only the MDV MEQ gene was shown to have transforming properties. Further evidence that MEQ is probably the principal oncogene in MDV came when researchers used overlapping cosmid clones of MDV and demonstrated that deleting MEQ resulted in a highly protective Marek's disease (MD) vaccine. We deleted both copies of MEQ from a bacterial artificial chromosome clone (BAC) of MDV. The virus, BACdelMEQ, was completely attenuated and did not appear to have any adverse effect on chicken body weight in MDV maternal-antibody-positive chickens, as measured at 8 wk of age. In two protection studies, BACdelMEQ efficiently protected susceptible chickens from a challenge by MDV strain 686, one of the most virulent MDV strains. In both protection studies, the BACdelMEQ protected chickens significantly better than the commercial MD vaccine, CVI988/Rispens. Only the protein-coding sequences of MEQ were deleted and all upstream and downstream regulatory sequences were left intact. Thus, BACdelMEQ has the potential to be a superior MD vaccine as well as a vector to deliver various foreign genes to poultry.

A small, variable, and irregular killer cell Ig-like receptor locus accompanies the absence of MHC-C and MHC-G in gibbons.

The killer cell Ig-like receptors (KIRs) of NK cells recognize MHC class I ligands and function in placental reproduction and immune defense against pathogens. During the evolution of monkeys, great apes, and humans, an ancestral KIR3DL gene expanded to become a diverse and rapidly evolving gene family of four KIR lineages. Characterizing the KIR locus are three framework regions, defining two intervals of variable gene content. By analysis of four KIR haplotypes from two species of gibbon, we find that the smaller apes do not conform to these rules. Although diverse and irregular in structure, the gibbon haplotypes are unusually small, containing only two to five functional genes. Comparison with the predicted ancestral hominoid KIR haplotype indicates that modern gibbon KIR haplotypes were formed by a series of deletion events, which created new hybrid genes as well as eliminating ancestral genes. Of the three framework regions, only KIR3DL3 (lineage V), defining the 5' end of the KIR locus, is present and intact on all gibbon KIR haplotypes. KIR2DL4 (lineage I) defining the central framework region has been a major target for elimination or inactivation, correlating with the absence of its putative ligand, MHC-G, in gibbons. Similarly, the MHC-C-driven expansion of lineage III KIR genes in great apes has not occurred in gibbons because they lack MHC-C. Our results indicate that the selective forces shaping the size and organization of the gibbon KIR locus differed from those acting upon the KIR of other hominoid species.

BAC to immunology--bacterial artificial chromosome-mediated transgenesis for targeting of immune cells.

Thirty years after the first transgenic mouse was produced, a plethora of genetic tools has been developed to study immune cells in vivo. A powerful development is the bacterial artificial chromosome (BAC) transgenic approach, combining advantages of both conventional transgenic and knock-in gene-targeting strategies. In immunology the potential of BAC transgenic technology has yet to be fully harvested and, combined with a variety of elegant genetic tools, it will allow the analysis of complex immunological processes in vivo. In this short review, we discuss the applications of BACs in immunology, such as identification of regulatory regions, expression and cell-fate mapping, cell ablation, conditional mutations and the generation of humanized mice.

DNA immunization with a herpes simplex virus 2 bacterial artificial chromosome.

Construction of a herpes simplex virus 2 (HSV-2) bacterial artificial chromosome (BAC) is described. BAC vector sequences were inserted into the thymidine kinase gene of HSV-2 by homologous recombination. DNA from cells infected with the resulting recombinant virus was transformed into E. coli, and colonies containing the HSV-2 BAC (HSV2-BAC) were isolated and analyzed for the expected genotype. HSV2-BAC DNA was infectious when transfected back into mammalian cells and the resulting virus was thymidine kinase negative. When used to immunize mice, the HSV2-BAC DNA elicited a strong HSV-2 specific antibody response that was equal to or greater than live virus immunization. Further, HSV2-BAC immunization was protective when animals were challenged with a lethal dose of virus. The utility of the HSV2-BAC for construction of recombinant virus genomes was demonstrated by elimination of the HSV-2 glycoprotein D (gD) gene. A recombinant HSV-2 BAC with the gD gene deleted was isolated and shown to be incapable of producing infectious virus following transfection unless an HSV gD gene was expressed in a complementing cell line. Immunization of mice with the HSV2 gD-BAC also elicited an HSV-2 specific antibody response and was protective. The results demonstrate the feasibility of DNA immunization with HSV-2 bacterial artificial chromosomes for replicating and nonreplicating candidate HSV-2 vaccines, as well as the utility of BAC technology for construction and maintenance of novel HSV-2 vaccines. The results further suggest that such technology will be a powerful tool for dissecting the immune response to HSV-2.