Single nucleotide polymorphism detection by polymerase chain reaction-restriction fragment length polymorphism

ArticleNATURE PROTOCOLS. 2(11): 2857-2864 (2007)journal articl

Compound Evolutionary History of the Rhesus Macaque Mhc Class I B Region Revealed by Microsatellite Analysis and Localization of Retroviral Sequences

In humans, the single polymorphic B locus of the major histocompatibility complex is linked to the microsatellite MIB. In rhesus macaques, however, haplotypes are characterized by the presence of unique combinations of multiple B genes, which may display different levels of polymorphism. The aim of the study was to shed light on the evolutionary history of this highly complex region. First, the robustness of the microsatellite MIB-linked to almost half of the B genes in rhesus macaques (Mamu-B)–for accurate B haplotyping was studied. Based on the physical map of an established haplotype comprising 7 MIB loci, each located next to a certain Mamu-B gene, two MIB loci, MIB1 and MIB6, were investigated in a panel of MHC homozygous monkeys. MIB1 revealed a complex genotyping pattern, whereas MIB6 analysis resulted in the detection of one or no amplicon. Both patterns are specific for a given B haplotype, show Mendelian segregation, and even allow a more precise haplotype definition than do traditional typing methods. Second, a search was performed for retroelements that may have played a role in duplication processes as observed in the macaque B region. This resulted in the description of two types of duplicons. One basic unit comprises an expressed Mamu-B gene, adjacent to an HERV16 copy closely linked to MIB. The second type of duplicon comprises a Mamu-B (pseudo)gene, linked to a truncated HERV16 structure lacking its MIB segment. Such truncation seems to coincide with the loss of B gene transcription. Subsequent to the duplication processes, recombination between MIB and Mamu-B loci appears to have occurred, resulting in a hyperplastic B region. Thus, analysis of MIB in addition to B loci allows deciphering of the compound evolutionary history of the class I B region in Old World monkeys

Genomic plasticity of the MHC class I A region in rhesus macaques: extensive haplotype diversity at the population level as revealed by microsatellites

The Mamu-A genes of the rhesus macaque show different degrees of polymorphism, transcription level variation, and differential haplotype distribution. Per haplotype, usually one “major” transcribed gene is present, A1 (A7), in various combinations with “minor” genes, A2 to A6. In silico analysis of the physical map of a heterozygous animal revealed the presence of similar Mamu-A regions consisting of four duplication units, but with dissimilar positions of the A1 genes on both haplotypes, and in combination with different minor genes. Two microsatellites, D6S2854 and D6S2859, have been selected as potential tools to characterize this complex region. Subsequent analysis of a large breeding colony resulted in the description of highly discriminative patterns, displaying copy number variation in concert with microsatellite repeat length differences. Sequencing and segregation analyses revealed that these patterns are unique for each Mamu-A haplotype. In animals of Indian, Burmese, and Chinese origin, 19, 15, or 9 haplotypes, respectively, could be defined, illustrating the occurrence of differential block duplications and subsequent rearrangements by recombination. The haplotypes can be assigned to 12 unique combinations of genes (region configurations). Although most configurations harbor two transcribed A genes, one or three genes per haplotype are also present. Additionally, haplotypes lacking an A1 gene or with an A1 duplication appear to exist. The presence of different transcribed A genes/alleles in monkeys from various origins may have an impact on differential disease susceptibilities. The high-throughput microsatellite technique will be a valuable tool in animal selection for diverse biomedical research projects

Rhesus macaque MHC class I molecules show differential subcellular localizations

The MHC class I gene family of rhesus macaques is characterised by considerable gene duplications. While a HLA-C-orthologous gene is absent, the Mamu-A and in particular the Mamu-B genes have expanded, giving rise to plastic haplotypes with differential gene content. Although some of the rhesus macaque MHC class I genes are known to be associated with susceptibility/resistance to infectious diseases, the functional significance of duplicated Mamu-A and Mamu-B genes and the expression pattern of their encoded proteins are largely unknown. Here, we present data of the subcellular localization of AcGFP-tagged Mamu-A and Mamu-B molecules. We found strong cell surface and low intracellular expression for Mamu-A1, Mamu-A2 and Mamu-A3-encoded molecules as well as for Mamu-B*01704, Mamu-B*02101, Mamu-B*04801, Mamu-B*06002 and Mamu-B*13401. In contrast, weak cell surface and strong intracellular expression was seen for Mamu-A4*1403, Mamu-B*01202, Mamu-B*02804, Mamu-B*03002, Mamu-B*05704, Mamu-I*010201 and Mamu-I*0121. The different expression patterns were assigned to the antigen-binding α1 and α2 domains, suggesting failure of peptide binding is responsible for retaining ‘intracellular’ Mamu class I molecules in the endoplasmic reticulum. These findings indicate a diverse functional role of the duplicated rhesus macaque MHC class I genes

S100A7 and the progression of breast cancer

The S100 gene family comprises more than 20 members whose protein sequences encompass at least one EF-hand Ca(2+ )binding motif. The expression of individual family members is not ubiquitous for all tissues and there appears to be an element of tissue-specific expression. Molecular analysis of breast tumors has revealed that several S100s, including S100A2, S100A4 and S100A7, exhibit altered expression levels during breast tumorigenesis and/or progression. Subsequent studies have started to describe a functional role for these S100 proteins as well as their mechanism of action and the biochemical pathways they modify. The present review outlines what is known about S100A7 in breast cancer and summarizes the need to better understand the importance of this protein in breast cancer

MHC class I A region diversity and polymorphism in macaque species

The HLA-A locus represents a single copy gene that displays abundant allelic polymorphism in the human population, whereas, in contrast, a nonhuman primate species such as the rhesus macaque (Macaca mulatta) possesses multiple HLA-A-like (Mamu-A) genes, which parade varying degrees of polymorphism. The number and combination of transcribed Mamu-A genes present per chromosome display diversity in a population of Indian animals. At present, it is not clearly understood whether these different A region configurations are evolutionarily stable entities. To shed light on this issue, rhesus macaques from a Chinese population and a panel of cynomolgus monkeys (Macaca fascicularis) were screened for various A region-linked variations. Comparisons demonstrated that most A region configurations are old entities predating macaque speciation, whereas most allelic variation (>95%) is of more recent origin. The latter situation contrasts the observations of the major histocompatibility complex class II genes in rhesus and cynomolgus macaques, which share a high number of identical alleles (>30%) as defined by exon 2 sequencing

Signal Peptide-Dependent Inhibition of MHC Class I Heavy Chain Translation by Rhesus Cytomegalovirus

The US2-11 region of human and rhesus cytomegalovirus encodes a conserved family of glycoproteins that inhibit MHC-I assembly with viral peptides, thus preventing cytotoxic T cell recognition. Since HCMV lacking US2-11 is no longer able to block assembly and transport of MHC-I, we examined whether this is also observed for RhCMV lacking the corresponding region. Unexpectedly, recombinant RhCMV lacking US2-11 was still able to inhibit MHC-I expression in infected fibroblasts, suggesting the presence of an additional MHC-I evasion mechanism. Progressive deletion analysis of RhCMV-specific genomic regions revealed that MHC-I expression is fully restored upon additional deletion of rh178. The protein encoded by this RhCMV-specific open reading frame is anchored in the endoplasmic reticulum membrane. In the presence of rh178, RhCMV prevented MHC-I heavy chain (HC) expression, but did not inhibit mRNA transcription or association of HC mRNA with translating ribosomes. Proteasome inhibitors stabilized a HC degradation intermediate in the absence of rh178, but not in its presence, suggesting that rh178 prevents completion of HC translation. This interference was signal sequence-dependent since replacing the signal peptide with that of CD4 or murine HC rendered human HCs resistant to rh178. We have identified an inhibitor of antigen presentation encoded by rhesus cytomegalovirus unique in both its lack of homology to any other known protein and in its mechanism of action. By preventing signal sequence-dependent HC translocation, rh178 acts prior to US2, US3 and US11 which attack MHC-I proteins after protein synthesis is completed. Rh178 is the first viral protein known to interfere at this step of the MHC-I pathway, thus taking advantage of the conserved nature of HC leader peptides, and represents a new mechanism of translational interference

A snapshot of the Mamu-B genes and their allelic repertoire in rhesus macaques of Chinese origin

The major histocompatibility complex class I gene repertoire was investigated in a large panel of rhesus macaques of Chinese origin. As observed in Indian animals, subjects of Chinese derivation display Mamu-B gene copy number variation, and the sum of expressed genes varies among haplotypes. In addition, these genes display differential transcription levels. The majority of the Mamu-B alleles discovered during this investigation appear to be unique for the population studied. Only one particular Mamu-B haplotype is shared between Indian and Chinese animals, and it must have been present in the progenitor stock. Hence, the data highlight the fact that most allelic polymorphism, and most of the Mamu-B haplotypes themselves, are of relatively recent origin and were most likely generated after the separation of the Indian and Chinese rhesus macaque populations

Genome-Wide Expression Profiling of Five Mouse Models Identifies Similarities and Differences with Human Psoriasis

Development of a suitable mouse model would facilitate the investigation of pathomechanisms underlying human psoriasis and would also assist in development of therapeutic treatments

Zebrafish brd2a and brd2b are paralogous members of the bromodomain-ET (BET) family of transcriptional coregulators that show structural and expression divergence

<p>Abstract</p> <p>Background</p> <p>Brd2 belongs to the bromodomain-extraterminal domain (BET) family of transcriptional co-regulators, and functions as a pivotal histone-directed recruitment scaffold in chromatin modification complexes affecting signal-dependent transcription. Brd2 facilitates expression of genes promoting proliferation and is implicated in apoptosis and in egg maturation and meiotic competence in mammals; it is also a susceptibility gene for juvenile myoclonic epilepsy (JME) in humans. The <it>brd2 </it>ortholog in <it>Drosophila </it>is a maternal effect, embryonic lethal gene that regulates several homeotic loci, including Ultrabithorax. Despite its importance, there are few systematic studies of <it>Brd2 </it>developmental expression in any organism. To help elucidate both conserved and novel gene functions, we cloned and characterized expression of <it>brd2 </it>cDNAs in zebrafish, a vertebrate system useful for genetic analysis of development and disease, and for study of the evolution of gene families and functional diversity in chordates.</p> <p>Results</p> <p>We identify cDNAs representing two paralogous <it>brd2 </it>loci in zebrafish, <it>brd2a </it>on chromosome 19 and <it>brd2b </it>on chromosome 16. By sequence similarity, syntenic and phylogenetic analyses, we present evidence for structural divergence of <it>brd2 </it>after gene duplication in fishes. <it>brd2 </it>paralogs show potential for modular domain combinations, and exhibit distinct RNA expression patterns throughout development. RNA <it>in situ </it>hybridizations in oocytes and embryos implicate <it>brd2a </it>and <it>brd2b </it>as maternal effect genes involved in egg polarity and egg to embryo transition, and as zygotic genes important for development of the vertebrate nervous system and for morphogenesis and differentiation of the digestive tract. Patterns of <it>brd2 </it>developmental expression in zebrafish are consistent with its proposed role in <it>Homeobox </it>gene regulation.</p> <p>Conclusion</p> <p>Expression profiles of zebrafish <it>brd2 </it>paralogs support a role in vertebrate developmental patterning and morphogenesis. Our study uncovers both maternal and zygotic contributions of <it>brd2</it>, the analysis of which may provide insight into the earliest events in vertebrate development, and the etiology of some forms of epilepsy, for which zebrafish is an important model. Knockdowns of <it>brd2 </it>paralogs in zebrafish may now test proposed function and interaction with homeotic loci in vertebrates, and help reveal the extent to which functional novelty or partitioning has occurred after gene duplication.</p