Genetic analysis of Dengue 3 virus subtype III 5' and 3' non-coding regions

The emergence of dengue hemorrhagic fever in Sri Lanka in 1989 correlated with the appearance of a genetic variant of Dengue 3 virus (DENV-3) subtype III (group B), closely related to the endemic group A variant. We studied the 5' and 3' non-coding regions (NCRs) of 15 DENV-3 subtype III isolates from Sri Lanka, Nicaragua and Martinique and found variability in the 3' NCRs. This included an 11-nucleotide insertion common to all isolates examined and two nucleotide differences that segregated viruses geographically into an American and a Sri Lankan group. Comparisons also identified three nucleotide differences shared by all group A Sri Lankan DENV-3 III isolates linked to mild disease epidemics but not group B Sri Lankan and group B-associated American isolates linked to severe disease epidemics. Clustering of the Latin American/Caribbean isolates with Sri Lankan group B DENV-3 in phylogenetic analyses supports the proposed common East African origin for all these strains and confirms the use of the 3' NCR for molecular epidemiologic studies of DENV-3. The differences in the 3' NCRs reported here, as well as potential alterations in the structural and non-structural coding genes, may contribute to the increased pathogenicity of group B DENV-3.     

Initiation of pre-B cell receptor signaling: common and distinctive features in human and mouse

B cell development in the bone marrow is a highly regulated process and expression of a functional pre-BCR represents a crucial checkpoint, common to human and mouse. In this review, we discuss pre-BCR analogies and differences between the two species leading to pre-B cell differentiation and proliferation. In addition, the mechanisms triggering pre-BCR activation are reviewed, taking into account the recent report of heparan sulfates and galectin 1 as stromal cell-derived pre-BCR ligands. Finally, ligand-induced pre-BCR activation models are proposed on the bases of the differences reported for pre-BCR and IL7 dependencies in the two species.     

Association between the Igk and Igh immunoglobulin loci mediated by the 3' Igk enhancer induces 'decontraction' of the Igh locus in pre-B cells

Variable-(diversity)-joining (V(D)J) recombination at loci encoding the immunoglobulin heavy chain (Igh) and immunoglobulin light chain (Igk) takes place sequentially during successive stages in B cell development. Using three-dimensional DNA fluorescence in situ hybridization, here we identify a lineage-specific and stage-specific interchromosomal association between these two loci that marks the transition between Igh and Igk recombination. Colocalization occurred between pericentromerically located alleles in pre-B cells and was mediated by the 3' Igk enhancer. Deletion of this regulatory element prevented association of the Igh and Igk loci, inhibited pericentromeric recruitment and locus 'decontraction' of an Igh allele, and resulted in greater distal rearrangement of the gene encoding the variable heavy-chain region. Our data indicate involvement of the Igk locus and its 3' enhancer in directing the Igh locus to a repressive nuclear subcompartment and inducing the Igh locus to decontract.     

Expression of CD103 identifies human regulatory T-cell subsets

BACKGROUND: Analysis of naturally occurring T regulatory CD4+ (nTreg) cells in human diseases is hampered by the lack of specific surface marker. Indeed, the CD25 antigen, which is typically used to identify nTreg cells, is also expressed on activated effector T cells. OBJECTIVE: We sought to examine whether CD4+ T cells bearing CD103 are suppressor cells, regardless of CD25 coexpression. METHODS: We first compared freshly isolated tonsillar CD103+ CD25- cells with their CD103- CD25high counterparts for their capacity to suppress T-cell response and their expression of FoxP3 mRNA. Next CD103 was induced on neonatal or adult CD4+ T cells stimulated with allogeneic dendritic cells, and the CD103+ and CD103- fractions were compared as above. RESULTS: Tonsillar CD4+ CD103+ CD25- T cells displayed comparable suppressive activity and contained similar amounts of FoxP3 mRNA as their CD103- CD25high counterparts. In vitro-generated alloantigen-primed CD103+ cells coexpressed CD25, suppressed T-cell activation, and contained more FoxP3 mRNA than the CD103- CD25+ cells isolated from the same cultures. Finally, neonatal alloreactive cells contained more CD103+ Treg cells than their adult counterparts and, unlike the latter, became hyporesponsive to the priming alloantigens. CONCLUSIONS: The examination of CD103 and CD25 coexpression allows identification of 3 subsets of human CD4+ nTreg cells, and the detection of CD103 on CD4+ T cells identifies nTreg cells, regardless of CD25 coexpression. CLINICAL IMPLICATIONS: The greater induction of CD103+ suppressor cells by cord blood should be related to its successful clinical use as an alternative to adult bone marrow transplantation.     

Polymorphism in the 5' upstream regulatory and 3' untranslated regions of the HLA-G gene in relation to soluble HLA-G and IL-10 expression

The nonclassical human leukocyte antigen (HLA) class Ib gene HLA-G may be important for the induction and maintenance of immune tolerance between the mother and the semi-allogeneic fetus during pregnancy. Expression of HLA-G can influence cytokine and cytotoxic T-lymphocyte responses. Different HLA-G mRNA isoform expression patterns have been associated with HLA-G polymorphism, especially with a 14-bp insertion deletion polymorphism in the 3' untranslated region (3'UTR) of the HLA-G gene. A significantly high level of interleukin-10 (IL-10) secretion is observed in homozygous +14/+14-bp HLA-G peripheral blood mononuclear cells after lipopolysaccharide (LPS) stimulation. This study finds that polymorphism in the 5' upstream regulatory region (5'URR) of the HLA-G gene may also be implicated in differences in IL-10 secretion. However, this may also be due to linkage disequilibrium with the 14-bp polymorphism. A single-nucleotide polymorphism located -477 bp from the start site of exon 1 had a significant association with IL-10 concentrations but not after correction (p=0.011; pc=0.154). This polymorphism is located next to a heat shock element. Eighteen 5'-URR/3'-UTR HLA-G haplotypes were defined; one common homozygous genotype based on these haplotypes was significantly associated with a high IL-10 level after LPS stimulation compared to certain other genotypes. This study indicates that polymorphism in the 5'-URR of the HLA-G gene may have functional significance, although a new line of investigations is needed to elucidate these findings.     

Comparative DNA sequence analysis of mouse and human protocadherin gene clusters

The genomic organization of the human protocadherin alpha, beta, and gamma gene clusters (designated Pcdh alpha [gene symbol PCDHA], Pcdh beta [PCDHB], and Pcdh gamma [PCDHG]) is remarkably similar to that of immunoglobulin and T-cell receptor genes. The extracellular and transmembrane domains of each protocadherin protein are encoded by an unusually large "variable" region exon, while the intracellular domains are encoded by three small "constant" region exons located downstream from a tandem array of variable region exons. Here we report the results of a comparative DNA sequence analysis of the orthologous human (750 kb) and mouse (900 kb) protocadherin gene clusters. The organization of Pcdh alpha and Pcdh gamma gene clusters in the two species is virtually identical, whereas the mouse Pcdh beta gene cluster is larger and contains more genes than the human Pcdh beta gene cluster. We identified conserved DNA sequences upstream of the variable region exons, and found that these sequences are more conserved between orthologs than between paralogs. Within this region, there is a highly conserved DNA sequence motif located at about the same position upstream of the translation start codon of each variable region exon. In addition, the variable region of each gene cluster contains a rich array of CpG islands, whose location corresponds to the position of each variable region exon. These observations are consistent with the proposal that the expression of each variable region exon is regulated by a distinct promoter, which is highly conserved between orthologous variable region exons in mouse and human.     

Functional analysis of different regions of the positive-acting CYS3 regulatory protein of Neurospora crassa

In the filamentous fungus Neurospora crassa during conditions of sulfur limitation, CYS3, a major positive-acting regulatory protein, turns on the expression of an entire set of genes which encode permeases and enzymes involved in the acquisition of sulfur from environmental sources. CYS3 functions as a homodimeric protein and possesses a b-Zip domain that confers sequence-specific DNA binding. Expression of various hybrid GAL4-CYS3 fusion proteins in yeast was used to detect regions involved in gene activation. An amino-terminal serine/threonine-rich domain of CYS3 alone strongly activated expression of β-galactosidase, the yeast reporter. Moreover, mutant CYS3 proteins with amino-acid substitutions in this region that showed increased expression in Neurospora also displayed an enhanced activation potential in yeast. The cys-3 gene of the exotic N. crassa Mauriceville strain and of N. intermedia were cloned and demonstrated to be functional for gene activation and for sulfur-mediated regulation by complementation of a loss-of-function cys-3 mutation. The amino-terminal serine/threonine-rich region is highly conserved in these two CYS3 proteins, in agreement with the possibility that it serves as the activation domain. Surprisingly, an extended promoter region of the cys-3 gene in the Mauriceville strain and in N. intermedia was very well conserved with that of the standard N. crassa gene, including the presence of three CYS3-binding sites possibly involved in autogenous control. Results are presented which indicate that synthesis of the CYS3 regulatory protein is highly regulated and can be detected in the nucleus of cells subjected to sulfur de-repression, but is not found in the nucleus or the cytoplasm of S-repressed cells. The amino-acid substitutions of the CYS3 protein present in a temperature-sensitive cys-3 mutant and in a second-site revertant of a cys-3 null mutation are presented and are shown to affect their DNA-binding activities. Received: 9 January / 5 March 1998     

In a model of immunoglobulin heavy-chain (IGH)/MYC translocation, the Igh 3' regulatory region induces MYC expression at the immature stage of B cell development

Reciprocal translocations involving the immunoglobulin loci and the cellular oncogene MYC are hallmark mutations of the human postgerminal center B cell neoplasm, Burkitt's lymphoma. They are occasionally found in other B cell lymphomas, as well. Translocations involving the heavy chain locus (IGH) place the MYC gene either in cis with both the intronic enhancer Emu and the IGH 3' regulatory region (3'RR) or in cis with only the 3'RR. The result is deregulated MYC expression. Recent studies have led to some controversy as to when, during B lymphocyte development, IGH/MYC chromosome translocations take place. A related issue, relevant not only to lymphoma development but also to normal controls on IGH gene expression, is the stage, during B lymphocyte development, at which the 3'RR is capable of activating MYC expression. We have developed mice transgenic for a human MYC (hMYC) gene under control of the four core enhancers from the mouse Igh 3'RR. Unlike other transgenic mouse models where premature and inappropriate MYC expression disrupts normal B cell development, the hMYC transgene in these studies carries a mutation that prohibits MYC protein synthesis. As a result, hMYC expression can be analyzed in all of the normal B cell compartments. Our data show that hMYC is expressed almost exclusively in B-lineage cells and is induced to high levels as soon as bone marrow cells reach the immature B cell stage.     

Comparative DNA sequence analysis of mouse and human CC chemokine gene clusters.

The CC chemokines are a closely related subfamily of the chemokine superfamily. Most of the CC chemokine genes form a cluster on chromosome 11 in mice and chromosome 17 in humans. To date, 11 and 16 functional genes have been localized within the mouse and human clusters, respectively. Notably, some of the genes within these clusters appear to have no counterparts between the two species, and the orthologous relationships of some of the genes are difficult to establish solely on the basis of amino acid similarity. In this study, we have taken a comparative genomic approach to reveal some of the features that may be involved in the dynamic evolution of these gene clusters. We sequenced a 122-kb region containing five chemokine genes of the mouse CC cluster. This mouse sequence was combined with those determined by the Mouse Genome Sequencing Project, and the entire sequence of the mouse CC cluster was compared with that of the corresponding cluster in the human genome by percent identity plot and dot-plot analyses. Although no additional chemokine genes have been found in these clusters, our analysis has revealed that numerous gene rearrangements have occurred even after the diversification of rodents and primates, resulting in several species-specific chemokine genes and pseudogenes. In addition, phylogenetic analysis and comparison of the genomic sequences unambiguously identified the orthologous relationships of some of the chemokine genes in the mouse and human CC gene clusters.     

Comparative genome analysis of the mouse imprinted gene impact and its nonimprinted human homolog IMPACT: toward the structural basis for species-specific imprinting

Mouse Impact is a paternally expressed gene encoding an evolutionarily conserved protein of unknown function. Here we identified IMPACT, the human homolog of Impact, on chromosome 18q11. 2-12.1, a region syntenic to the mouse Impact locus. IMPACT was expressed biallelically in brain and in various tissues from two informative fetuses and in peripheral blood from an informative adult. To reveal the structural basis for the difference in allelic expression between the two species, we elucidated complete genome sequences for both mouse Impact ( approximately 38 kb) and human IMPACT ( approximately 30 kb). Sequence comparison revealed that the two genes share a well-conserved exon-intron organization but bear significantly different CpG islands. The mouse island lies in the first intron and contains characteristic tandem repeats. Furthermore, this island serves as a differentially methylated region (DMR) consisting of a hypermethylated maternal allele and an unmethylated paternal allele. Intriguingly, this intronic island is missing from the nonimprinted human IMPACT, whose sole CpG island spans the first exon, lacks any apparent repeats, and escapes methylation on both chromosomes. These results suggest that the intronic DMR plays a role in the imprinting of Impact.     

Cellular proteins bind to the 3' and 5' untranslated regions of dengue 2 virus genome

In vitro generated cloned full length dengue 2 virus untranslated regions (UTRs) were used in RNA gel mobility shift assays to examine cellular factors binding to the virus genomes. Cellular factors in lysates of Vero (monkey) and C6/36 (mosquito) cells bound specifically and non-specifically to the dengue 2 virus 3' UTR. Non-specific interaction with the 5' UTR, resulting in formation of at least 4 band shift complexes was noted with lysate of the C6/36 cells only. Pre-treating the cell lysates with proteinase K affected binding of cellular factors to the dengue 2 virus UTRs, suggesting that the cellular factors were proteins. These findings suggest that cellular proteins could interact with specific sites on the dengue virus genomes.     

Quantitative proteomic analysis of purified yeast kinetochores identifies a PP1 regulatory subunit

The kinetochore is a macromolecular complex that controls chromosome segregation and cell cycle progression. When sister kinetochores make bioriented attachments to microtubules from opposite poles, the spindle checkpoint is silenced. Biorientation and the spindle checkpoint are regulated by a balance between the Ipl1/Aurora B protein kinase and the opposing activity of protein phosphatase I (PP1). However, little is known about the regulation of PP1 localization and activity at the kinetochore. Here, we developed a method to purify centromere-bound kinetochores and used quantitative proteomics to identify the Fin1 protein as a PP1 regulatory subunit. The Fin1/PP1 complex is regulated by phosphorylation and 14–3–3 protein binding. When Fin1 is mislocalized, bipolar spindles fail to assemble but the spindle checkpoint is inappropriately silenced due to PP1 activity. These data suggest that Fin1 is a PP1 regulatory subunit whose spatial and temporal activity must be precisely controlled to ensure genomic stability.     

Phylogenetic analysis of Brazilian Flavivirus using nucleotide sequences of parts of NS5 gene and 3' non-coding regions

Viruses of the genus Flavivirus, which are arboviruses, of the Flaviviridae family, are amongst the most important agents of infectious disease in Brazil, causing human infections with a high morbility and mortality. In this work, the phylogeny of 14 virus amplicon sequences that were obtained by RT-PCR with universal primers for mosquito-borne Flavivirus were studied. The amplicons included a region of the Flavivirus genome of 129 nucleotides at the 3' terminus of the NS5 gene and the 145 initial nucleotides of the 3' non-coding region (NS5-3'NCR). Based on phylogenetic trees, most Brazilian Flaviviruses were grouped into two main branches, including a yellow-fever branch and a second main branch divided into a dengue branch that in its turn is subdivided into serotype 1, 2 and 4 branches, and another (Japanese Encephalitis Virus Complex) branch including SLE and Ilhéus. Rocio and Cacipacoré viruses were included in the Japanese Encephalitis Virus Complex branch in one of the two phylogenetic trees. Iguape virus appears in phylogenetic trees as a separate distant branch.