Tob-mediated cross-talk between MARCKS phosphorylation and ErbB-2 activation

The biochemical path for the activation of ErbB-2 by PKC activator was investigated in MDA-MB-231 human breast cancer cells. We found that PMA-induced phosphorylation of myristoylated alanine-rich C kinase substrate (MARCKS) increased its binding with Tob that exerts an anti-proliferative effect through the binding with ErbB-2. The phosphorylation site domain (PSD) of MARCKS was relevant to its interaction with Tob. Decreased binding of Tob with ErbB-2 and subsequent activation of ErbB-2 were observed in MDA-MB-231 cells in response to PMA treatment. The present study proposes that MARCKS phosphorylation by PKC removes Tob from ErbB-2 by increasing its binding affinity with Tob, and thereby activates the ErbB-2 mediated signal transduction.     

Nucleotide diversity on the ovine Y chromosome

To investigate the impact of male-mediated introgression during the evolution of sheep breeds, a sequencing approach was used to identify single nucleotide polymorphisms (SNPs) from the male-specific region of the ovine Y chromosome (MSY). A total of 4380 bp, which comprised nine fragments from five MSY genes was sequenced within a panel of 14 males from seven breeds. Sequence alignment identified a single segregating site, an A/G SNP located approximately 1685 bp upstream of the ovine SRY gene. The resulting estimation of nucleotide diversity (piY = 0.90 +/- 0.50 x 10(-4)) falls towards the lower end of estimates from other species. This was compared with the nucleotide diversity estimated from the autosomal component of the genome. Sequence analysis of 2933 bp amplified from eight autosomal genes revealed a nucleotide diversity (piA = 2.15 +/- 0.27 x 10(-3)) higher than previously reported for sheep. Following adjustment for the contrasting influence of effective population size and a male biased mutation rate, comparison revealed that approximately 10% of the expected nucleotide diversity is present on the ovine Y chromosome.     

The molecular diversity of the methanogenic community in a hypereutrophic freshwater lake determined by PCR-RFLP

AIMS: To combine database-held sequence information with a programme of experimental molecular ecology to define the methanogenic community of a hypereutrophic lake by a PCR-restriction fragment length polymorphism (RFLP) analysis. METHODS AND RESULTS: Methanogen diversity in a hypereutrophic freshwater lake was analysed using 16S rDNA PCR-RFLP. Database-held 16S rRNA gene sequences for 76 diverse methanogens were analysed for specific restriction sites that permitted unequivocal differentiation of methanogens. Restriction digestion and agarose gel electrophoresis of the 16S rDNA from selected methanogen pure cultures generated observed restriction profiles that corroborated the expected patterns. This method was then tested by analysing methanogen diversity in samples obtained over 1 year from sediment and water samples taken from the same sampling site. CONCLUSIONS: Restriction analysis of the 16S rRNA gene sequences from 157 methanogen clones generated from lakewater and sediment samples showed that over 50% were similar to Methanoculleus spp. Furthermore, a total of 16 RFLP types (1-16) were identified, eight of which contained no cultured representative archaeal 16S rRNA gene sequences. SIGNIFICANCE AND IMPACT OF THE STUDY: This RFLP strategy provides a robust and reliable means to rapidly identify methanogens in the environment.     

Behavioral and ecological correlates of foureye butterflyfish, Chaetodon capistratus, (Perciformes: Chaetodontidae) infected with Anilocra chaetodontis (Isopoda: Cymothoidae)

We observed the behavior and ecology of Chaetodon capistratus infected and uninfected with the ectoparasitic isopod Anilocra chaetodontis to assess whether there may be parasite induced alterations in host biology, host defenses against infection, and/or pathology related to infection. We also examined habitat related differences in infection rates. Infected fish had higher rates of interaction with conspecifics and spent more time in low flow environments (which might improve transmission of juvenile parasites to new hosts). Butterfly fish without isopods were chased more frequently by damselfishes, fed more, and had larger territories. Time spent near conspecifics, and fish condition and gonadosomatic index did not vary between infected and uninfected fish. These results suggest that foureye butterfly fish behavior is altered by the isopod parasite in order for the isopods to more easily gain mates or transmit offspring to new hosts.     

NMR structure and mutagenesis of the third Bir domain of the inhibitor of apoptosis protein XIAP

The inhibitor of apoptosis proteins (IAPs) regulate the caspase family of cysteine proteases, which play an important role in the execution of programmed cell death. Human X-linked inhibitor of apoptosis protein (XIAP) is a potent inhibitor of caspases-3, -7, and -9. Here we show that the Bir3 domain is the minimal region of XIAP that is needed for potent caspase-9 inhibition. The three-dimensional structure of the Bir3 domain of XIAP, determined by NMR spectroscopy, resembles a classical zinc finger and consists of five alpha-helices, a three-stranded beta-sheet, and a zinc atom chelated to three cysteines and one histidine. The structure of the Bir3 domain is similar to that of the Bir2 domain of XIAP but differs from the previously determined structure of the Bir3 domain of MIHB. Based on site-directed mutagenesis, we have identified the regions of the Bir3 domain of XIAP that are important for inhibiting caspase-9. Despite the structural similarities of the Bir2 and Bir3 domain of XIAP, a different set of residues were found to be critical for inhibiting the individual caspases. These results suggest that XIAP inhibits caspase-3 and caspase-9 in a different manner.     

Survey of the fragile X syndrome CGG repeat and the short-tandem-repeat and single-nucleotide-polymorphism haplotypes in an African American population

Previous studies have shown that specific short-tandem-repeat (STR) and single-nucleotide-polymorphism (SNP)-based haplotypes within and among unaffected and fragile X white populations are found to be associated with specific CGG-repeat patterns. It has been hypothesized that these associations result from different mutational mechanisms, possibly influenced by the CGG structure and/or cis-acting factors. Alternatively, haplotype associations may result from the long mutational history of increasing instability. To understand the basis of the mutational process, we examined the CGG-repeat size, three flanking STR markers (DXS548-FRAXAC1-FRAXAC2), and one SNP (ATL1) spanning 150 kb around the CGG repeat in unaffected (n=637) and fragile X (n=63) African American populations and compared them with unaffected (n=721) and fragile X (n=102) white populations. Several important differences were found between the two ethnic groups. First, in contrast to that seen in the white population, no associations were observed among the African American intermediate or "predisposed" alleles (41-60 repeats). Second, two previously undescribed haplotypes accounted for the majority of the African American fragile X population. Third, a putative "protective" haplotype was not found among African Americans, whereas it was found among whites. Fourth, in contrast to that seen in whites, the SNP ATL1 was in linkage equilibrium among African Americans, and it did not add new information to the STR haplotypes. These data indicate that the STR- and SNP-based haplotype associations identified in whites probably reflect the mutational history of the expansion, rather than a mutational mechanism or pathway.     

Phylogenetic tests of the hypothesis of block duplication of homologous genes on human chromosomes 6, 9, and 1

There are 10 gene families that have members on both human chromosome 6 (6p21.3, the location of the human major histocompatibility complex [MHC]) and human chromosome 9 (mostly 9q33-34). Six of these families also have members on mouse chromosome 17 (the mouse MHC chromosome) and mouse chromosome 2. In addition, four of these families have members on human chromosome 1 (1q21-25 and 1p13), and two of these have members on mouse chromosome 1. One hypothesis to explain these patterns is that members of the 10 gene families of human chromosomes 6 and 9 were duplicated simultaneously as a result of polyploidization or duplication of a chromosome segment ("block duplication"). A subsequent block duplication has been proposed to account for the presence of representatives of four of these families on human chromosome 1. Phylogenetic analyses of the 9 gene families for which data were available decisively rejected the hypothesis of block duplication as an overall explanation of these patterns. Three to five of the genes on human chromosomes 6 and 9 probably duplicated simultaneously early in vertebrate history, prior to the divergence of jawed and jawless vertebrates, and shortly after that, all four of the genes on chromosomes 1 and 9 probably duplicated as a block. However, the other genes duplicated at different times scattered over at least 1.6 billion years. Since the occurrence of these clusters of related genes cannot be explained by block duplication, one alternative explanation is that they cluster together because of shared functional characteristics relating to expression patterns.      

Evolutionary distances for protein-coding sequences: modeling site- specific residue frequencies

Estimation of evolutionary distances from coding sequences must take into account protein-level selection to avoid relative underestimation of longer evolutionary distances. Current modeling of selection via site-to-site rate heterogeneity generally neglects another aspect of selection, namely position-specific amino acid frequencies. These frequencies determine the maximum dissimilarity expected for highly diverged but functionally and structurally conserved sequences, and hence are crucial for estimating long distances. We introduce a codon- level model of coding sequence evolution in which position-specific amino acid frequencies are free parameters. In our implementation, these are estimated from an alignment using methods described previously. We use simulations to demonstrate the importance and feasibility of modeling such behavior; our model produces linear distance estimates over a wide range of distances, while several alternative models underestimate long distances relative to short distances. Site-to-site differences in rates, as well as synonymous/nonsynonymous and first/second/third-codon-position differences, arise as a natural consequence of the site-to-site differences in amino acid frequencies.