Thomas William Beck

Functions, Structure, and Read-Through Alternative Splicing of Feline APOBEC3 Genes

BackgroundOver the past years a variety of host restriction genes have been identified in human and mammals that modulate retrovirus infectivity, replication, assembly, and/or cross-species transmission. Among these host-encoded restriction factors, the APOBEC3 (A3; apolipoprotein B mRNA-editing catalytic polypeptide 3) proteins are potent inhibitors of retroviruses and retrotransposons. While primates encode seven of these genes (A3A to A3H), rodents carry only a single A3 gene.ResultsHere we identified and characterized several A3 genes in the genome of domestic cat (Felis catus) by analyzing the genomic A3 locus. The cat genome presents one A3H gene and three very similar A3C genes (a-c), probably generated after two consecutive gene duplications. In addition to these four one-domain A3 proteins, a fifth A3, designated A3CH, is expressed by read-through alternative splicing. Specific feline A3 proteins selectively inactivated only defined genera of feline retroviruses: Bet-deficient feline foamy virus was mainly inactivated by feA3Ca, feA3Cb, and feA3Cc, while feA3H and feA3CH were only weakly active. The infectivity of Vif-deficient feline immunodeficiency virus and feline leukemia virus was reduced only by feA3H and feA3CH, but not by any of the feA3Cs. Within Felidae, A3C sequences show significant adaptive selection, but unexpectedly, the A3H sequences present more sites that are under purifying selection.ConclusionOur data support a complex evolutionary history of expansion, divergence, selection and individual extinction of antiviral A3 genes that parallels the early evolution of Placentalia, becoming more intricate in taxa in which the arms race between host and retroviruses is harsher.

Craf-1 protein kinase is essential for mouse development

The three mammalian Raf serine/threonine protein kinases mediate the transduction of proliferative and differentiative signals from a variety of cell surface receptors to the nucleus. We report here that Craf-1 is essential for mouse development, as its mutation results in embryonic lethality. Developmental defects are found in mutant placentas as well as in the skin and in the lungs of mutant embryos. Craf-1 mutants also display a generalized growth retardation which is consistent with the ubiquitous expression of Craf-1 and which could be due to the reduced proliferation of mutant cells. Interestingly, the time-point of embryonal death varies depending on the genetic background. This suggests that Craf-1-mediated signaling is affected by genetic background-specific alleles of other genes.

Sequences, Annotation and Single Nucleotide Polymorphism of the Major Histocompatibility Complex in the Domestic Cat

Two sequences of major histocompatibility complex (MHC) regions in the domestic cat, 2.976 and 0.362 Mbps, which were separated by an ancient chromosome break (55-80 MYA) and followed by a chromosomal inversion were annotated in detail. Gene annotation of this MHC was completed and identified 183 possible coding regions, 147 human homologues, possible functional genes and 36 pseudo/unidentified genes) by GENSCAN and BLASTN, BLASTP RepeatMasker programs. The first region spans 2.976 Mbp sequence, which encodes six classical class II antigens (three DRA and three DRB antigens) lacking the functional DP, DQ regions, nine antigen processing molecules (DOA/DOB, DMA/DMB, TAPASIN, and LMP2/LMP7,TAP1/TAP2), 52 class III genes, nineteen class I genes/gene fragments (FLAI-A to FLAI-S). Three class I genes (FLAI-H, I-K, I-E) may encode functional classical class I antigens based on deduced amino acid sequence and promoter structure. The second region spans 0.362 Mbp sequence encoding no class I genes and 18 cross-species conserved genes, excluding class I, II and their functionally related/associated genes, namely framework genes, including three olfactory receptor genes. One previously identified feline endogenous retrovirus, a baboon retrovirus derived sequence (ECE1) and two new endogenous retrovirus sequences, similar to brown bat endogenous retrovirus (FERVmlu1, FERVmlu2) were found within a 140 Kbp interval in the middle of class I region. MHC SNPs were examined based on comparisons of this BAC sequence and MHC homozygous 1.9x WGS sequences and found that 11,654 SNPs in 2.84 Mbp (0.00411 SNP per bp), which is 2.4 times higher rate than average heterozygous region in the WGS (0.0017 SNP per bp genome), and slightly higher than the SNP rate observed in human MHC (0.00337 SNP per bp).

The feline major histocompatibility complex is rearranged by an inversion with a breakpoint in the distal class I region

In order to determine the genomic organization of the major histocompatibility complex (MHC) of the domestic cat (Felis catus), DNA probes for 61 markers were designed from human MHC reference sequences and used to construct feline MHC BAC contig map spanning ARE1 in the class II region to the olfactory receptor complex in the extended class I region. Selected BAC clones were then used to identify feline-specific probes for the three regions of the mammalian MHC (class II–class III–class I) for radiation hybrid mapping and fluorescent in situ hybridization to refine the organization of the domestic cat MHC. The results not only confirmed that the p-arm of domestic cat B2 is inverted relative to human Chromosome 6, but also demonstrated that one inversion breakpoint localized to the distal segment of the MHC class I between TRIM39 and TRIM26. The inversion thus disjoined the ~2.85 Mb of MHC containing class II–class III–class I (proximal region) from the ~0.50 Mb of MHC class I/extended class I region, such that TRIM39 is adjacent to the Chromosome B2 centromere and TRIM26 is adjacent to the B2 telomere in the domestic cat.

Growth Factor Regulation of Cell Cycle Progression and Cell Fate Determination

The interaction between growth factors and their cell surface receptors initiates the cell cycle which under different physiological conditions can lead to cell growth, differentiation, survival or apoptosis (programmed cell death) (Figure 1). The sequence of events leading to cellular proliferation induced by growth factors involves activation of an intracellular signaling cascade resulting in induction of the expression of specific target genes in the nucleus which are sequentially and temporally expressed in defined classes, such as immediate early, delayed early and late. These gene products encode a large number of proteins which collaborate and indeed some are essential for driving cells through Gap1 (G1) of the cell cycle and into DNA synthesis (S phase). For successful entry into S phase, the growth factor must be in continuous contact with the cells. If however, the growth factor is removed prior to late G1 (R), cells will not commit to S phase and instead arrest in G1. This commitment step is controlled at least in part by certain members of a family of gene products, the cyclins, which are differentially regulated during the course of the cell cycle (Figure 1).

Genetic Markers for Oncogenes, Growth Factors, and Cystic Fibrosis

The techniques of molecular biology have had a dramatic effect on the advancement of human genetics. In particular, the development of restriction fragment length polymorphisms (RFLPs) has allowed researchers to generate genetic markers for virtually any region of the human genome. Most RFLPs occur when a mutation creates or deletes a recognition site for a restriction enzyme, generating a DNA fragment of altered size. In the simplest case this will create two alleles. A DNA probe which hybridizes to this fragment will detect the presence of these alleles in the DNA from different individuals. Probes used to detected RFLPs have been derived from both cloned genes and randomly isolated DNA segments. Thus, each RFLP is a genetically inherited marker for a precise location on a chromosome.

- Raf1: Raf1 protein kinase (vertebrates)

Raf1 is a serine/threonine-specific PK that functions in a signal transduction pathway(s) between the cell membrane and the nucleus. On mitogen stimulation of resting cells, Raf1 becomes hyperphosphorylated, which correlates with an increase of Raf1 kinase activity. Raf1 then phosphorylates and activates MAP kinase kinase (Mek), which in turn, activates MAP kinase (Erk1/2), leading to transactivation of transcription from promoters bearing AP-1 (Jun/Fos), Ets, and other response elements. Raf1 is required for the activation of c-Jun via phosphorylation in the transactivation domain. The enzyme is usually assayed in immune complexes as a mitogen-dependent transfer of radioactivity from [γ32P]ATP to Raf1 (autophosphorylation), histone type V-S, or any of several peptide substrates. The best assay for Raf1 activity is incubation with dephosphorylated MAP kinase kinase (Mek), which can then be assayed by phosphorylation of, or reactivation of, MAP kinase (Erkl/2). The recent cloning of Mek should allow direct assays of Raf1 phosphorylation. Raf1 mRNA is expressed in all human and mouse cell lines and all mouse tissues examined to date, as 3.1 and 3.4 kb RNAs in mouse and human, respectively. The levels of expression vary by as much as 5- to 10-fold between different mouse tissues. In mouse testes, Raf1 is expressed in both the somatic and germ cell compartments.

- A-Raf: A-Raf protein kinase (vertebrates)

A B-Raf oncogene was originally identified in an NIH3T3 cell transformant that was transfected with Ewing sarcoma DNA and a full-length B-Raf cDNA, and protein has since been characterized. It follows the same general functional rules and biochemical properties established for Raf1. NGF, EGF, or TPA stimulation of PC12 cells results in B-Raf becoming hyperphosphorylated, an increase of B-Raf kinase activity, and activation of the MAP kinase cascade. B-Raf is structurally similar to Raf1 and A-Raf but has a larger N-terminus. The CR2 region of B-Raf diverges slightly from this region in Raf1 and A-Raf. B-Raf has residues corresponding to all the major phosphorylation sites identified in Raf1. The enzyme, from PC12 cells, has been assayed in immune complexes as mitogen-dependent transfer of radioactivity from [γ32P]ATP to either B-Raf (autophosphorylation), casein, phosvitin, or a peptide substrate (B-Raf 359–386) as an immune complex. In contrast to Raf1, little or no mitogen-dependent stimulation of B-Raf kinase activity was observed with histone H1, histone H5, or syntide.