WASp in immune-system organization and function

The regulation of many immunological events depends on systems that mediate dynamic actin reorganization in response to signals from the cell membrane. The Wiskott-Aldrich syndrome protein (WASp) is the founding member of a family of proteins that have emerged as crucial effectors of Rho GTPases and activators of the cytoskeletal-organizing complex Arp2/3. Now, WASp has been shown to be intimately involved in many pathways that influence the function of the immune system. Disturbances in these systems result in the complex immunodysregulation of Wiskott-Aldrich syndrome.     

Nucleotide sequences of switch regions of immunoglobulin C epsilon and C gamma genes and their comparison

Immunoglobulin class switch involves a unique recombination event that takes place at the switch (S) region which is located 5' to each constant region (C) gene of the heavy (H) chain. For example, differentiation of the B lymphocyte from a mu-chain producer to an epsilon-chain producer is mediated by the switch recombination between the S mu and S epsilon regions. In order to elucidate the molecular mechanism for the switch recombination, we have determined nucleotide sequences surrounding the class switch recombination sites of the C epsilon and C gamma 3 genes and those in the 5' flanking regions of the C gamma 2a and C delta genes. The results indicate that the 5' flanking regions of all the CH genes except for the C delta gene contain the S regions which comprise tandem repetition of short unit sequences in agreement with the previous analyses of the S gamma 1, S gamma 2b, S mu, and S alpha regions. Comparison of the nucleotide sequences of all the S regions revealed that length as well as nucleotide sequences of the S regions vary among different classes of the CH gene, but they share short common sequences, (G)AGCT and TGGG(G). The nucleotide sequence of the S mu region is homologous to those of the other S regions in the decreasing order of the S epsilon, S alpha, S gamma 3, and (S gamma 1, S gamma 2b, s gamma 2a) regions. We have compared the nucleotide sequences immediately adjacent to the recombination sites of seven rearranged genes and have always fund tetranucleotides TGAG and/or TGGG, except for one case. Such tetranucleotides may constitute a part of the recognition sequence of a putative recombinase. These results provide further support for our previous proposal that the switch recombination may be facilitated by short common sequences dispersed in all the S regions.     

The immunoglobulin heavy chain switch: structural features of gamma 1 recombinant switch regions

The immunoglobulin heavy chain isotype switch is mediated by a DNA rearrangement involving specific genomic segments referred to as switch regions. Switch regions are composed of tandemly repeated simple sequences. The role of the tandemly repeated structure of switch regions in the switch recombination process is not understood. We mapped eight recombination sites--six in the gamma 1 and two in the gamma 3 tandem arrays. In addition, we obtained molecular clones representing three of the six gamma 1 rearrangements, and determined the nucleotide sequences of the recombination sites in each. In general, the rearrangements are confined to the tandem repeat units, and are not clustered in a particular portion of either the gamma 3 or gamma 1 switch region. Nucleotide sequence analysis of one of the recombinant clones, gamma M35, reveals evidence for a successive switch event wherein a recombination between S mu and S gamma 3 was followed by recombination 57 bp downstream with S gamma 1. gamma 1 sequence data from the molecular clones we obtained, together with similar data from other investigators regarding the gamma 1, gamma 2b, and gamma 2a switch regions, reveals that recombinations tend to occur at homologous positions of the respective gamma-unit repeats, adjacent to the elements AGCT and GGGG found in each. This finding suggests that the cutting and religation step of the recombination process is mediated by a recombinase common to the four gamma-isotypes.     

Genome-wide identification of runs of homozygosity islands and associated genes in local dairy cattle breeds

Runs of homozygosity (ROH) are widely used as predictors of whole-genome inbreeding levels in cattle. They identify regions that have an unfavorable effect on a phenotype when homozygous, but also identify the genes associated with traits of economic interest present in these regions. Here, the distribution of ROH islands and enriched genes within these regions in four dairy cattle breeds were investigated. Cinisara (71), Modicana (72), Reggiana (168) and Italian Holstein (96) individuals were genotyped using the 50K v2 Illumina BeadChip. The genomic regions most commonly associated with ROHs were identified by selecting the top 1% of the single nucleotide polymorphisms (SNPs) most commonly observed in the ROH of each breed. In total, 11 genomic regions were identified in Cinisara and Italian Holstein, and eight in Modicana and Reggiana, indicating an increased ROH frequency level. Generally, ROH islands differed between breeds. The most homozygous region (>45% of individuals with ROH) was found in Modicana on chromosome 6 within a quantitative trail locus affecting milk fat and protein concentrations. We identified between 126 and 347 genes within ROH islands, which are involved in multiple signaling and signal transduction pathways in a wide variety of biological processes. The gene ontology enrichment provided information on possible molecular functions, biological processes and cellular components under selection related to milk production, reproduction, immune response and resistance/susceptibility to infection and diseases. Thus, scanning the genome for ROH could be an alternative strategy to detect genomic regions and genes related to important economic traits.     

Evolutionary constraint in flanking regions of avian genes

An important comprehension from comparative genomic analysis is that sequence conservation beyond neutral expectations is frequently found outside protein-coding regions, indicating important functional roles of noncoding DNA. Understanding the causes of constraint on noncoding sequence evolution forms an important area of research, not least in light of the importance for understanding the evolution of gene expression. We aligned all orthologous genes of chicken and zebra finch together with 5 kb of their upstream and downstream noncoding sequences, to study the evolution of gene flanking sequences in the avian genome. Using ancestral repeats as a neutral reference, we detected significant evolutionary constraint in the 3' flanking region, highest directly after termination (60%) and then gradually decreasing to about 20% 5 kb downstream. Constraint was higher in annotated 3' untranslated regions (UTRs) than in non-UTRs at the same distance from the stop codon and higher in sequences annotated as microRNA (miRNA)-binding sites than in non-miRNA-binding sites within 3' UTRs. Constraint was also higher when estimated for a smaller data set of genes from more closely related songbird species, indicating turnover of functional elements during avian evolution. On the 5' flanking side constraint was readily seen within the first 125 bp immediately upstream of the start codon (34%) and was about 10% for remaining sequence within 5 kb upstream. Analysis of chicken polymorphism data gave further support for the highest constraint directly before and after the translated region. Finally, we found that genes evolving under the highest constraint measured by d(N)/d(S) also had the highest level of constraint in the 3' flanking region. This study broadens the insights into gene flanking sequence evolution by adding new findings from a vertebrate lineage other than mammals.     

Choice of base at silent codon site 3 is not selectively neutral in eucaryotic structural genes: It maintains excess short runs of weak and strong hydrogen bonding bases

On the average in the coding sequences of 30 eucaryotic structural genes the weak hydrogen bonding, W, (A or T) or strong hydrogen bonding, S, (C or G) base in codon site 3 was chosen to be unlike its neighbors on both sides up to two sites away. This preference produced the nonrandom excess of runs W and S of length one and two and the deficit of long runs observed earlier (Blaisdell 1982). The neighbors in the different codon, 3' to codon site 3, were as important in determining the choice as were the neighbors 5' in the same codon. Every amino acid except methionine and tryptophan, of least frequent occurrence, permits choice of W or S. The persistence of this preference could explain the observation that the rate of substitution of codon site 3 in functional genes is considerably less than in synonymous pseudo genes.     

Repeating sequences in promoter regions of eukaryotic genes]

Promoter regions of eukaryotic genes were analyzed for the presence of repeated fragments. It was found that the promotor sequences are abundant in direct, symmetrical, and inverted repeats. A computer system for searching and visualizing the repeats was developed.     

Identification of genes with fast-evolving regions in microbial genomes

Complete sequences of multiple strains of the same microbial species provide an invaluable source for studying the evolutionary dynamics between orthologous genes over a relatively short time scale. Usually the intensity of the selection pressure is inferred from a comparison between the nonsynonymous substitution rate and the synonymous substitution rate. In this paper, we propose an alternative method for detecting genes with one or more fast-evolving regions from pairwise comparisons of orthologous genes. Our method looks for regions with overrepresented nonsynonymous mutations along the alignment, and requires a higher nonsynonymous evolution rate in those regions than the neutral evolution rate. It identifies gene targets under intensive selection pressure that are not detected from the conventional rate comparison analysis. For those identified genes with known annotations, most of them have a clear role in processes such as bacterial defense and host–pathogen interactions. Gene sets reported from our method provide a measure of the phenotypic divergence between two closely related genomes.     

Periodicities in coding and noncoding regions of the genes

Gene population statistical studies of protein coding genes and introns have identified two types of periodicities on the purine/pyrimidine alphabet: (i) the modulo 3 periodicity or coding periodicity (periodicity P3) in protein coding genes of eukaryotes, prokaryotes, viruses, chloroplasts, mitochondria, plasmids and in introns of viruses and mitochondria, and (ii) the modulo 2 periodicity (periodicity P2) in the eukaryotic introns. The periodicity study is herein extended to the 5' and 3' regions of eukaryotes, prokaryotes and viruses and shows: (i) the periodicity P3 in the 5' and 3' regions of eukaryotes. Therefore, these observations suggest a unitary and dynamic concept for the genes as for a given genome, the 5' and 3' regions have the genetic information for protein coding genes and for introns: (1) In the eukaryotic genome, the 5' (P2 and P3) and 3' (P2 and P3) regions have the information for protein coding genes (P3) and for introns (P2). The intensity of P3 is high in 5' regions and weak in 3' regions, while the intensity of P2 is weak in 5' regions and high in 3' regions. (2) In the prokaryotic genome, the 5' (P3) and 3' (P3) regions have the information for protein coding genes (P3). (3) In the viral genome, the 5' (P3) and 3' (P3) regions have the information for protein coding genes (P3) and for introns (P3). The absence of P2 in viral introns (in opposition to eukaryotic introns) may be related to the absence of P2 in 5' and 3' regions of viruses.