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A novel evolutionary conserved human MOB gene of seven exons is predicted on the chromosome 10. MOB is supposed to express predominately in brain. At least three types of MOB transcripts are proposed. A protein encoded by MOB is five-pass transmembrane molecule.
Evolutionary conservation of homologous gene products from distantly related organisms provides an information resource of great value for elucidating protein structure and function. Sequence similarities also serve as molecular cross-references between diverse organisms that offer different, or complementary, experimental approaches for analyzing gene expression and biochemistry in normal and abnormal states. There are now countless examples of information about a protein from one species contributing to the understanding of biological phenomena or disease in another species. Such connections are often unanticipated and surprising, but there is an opportunity to make them more systematically as concerted genome sequencing projects progress. In the present review we focus on connections between yeast and human proteins and their functional implications. We present several 'case studies' as well as survey results derived from comprehensive sequence comparisons among all yeast and human proteins currently present in the public databases.
Hox genes encode for evolutionary conserved transcription factors that direct the proper development of the body plan. Despite decades of research, little is known regarding their downstream target genes, especially in vertebrates. The strong evolutionary conservation of their DNA-binding homeodomain, their generic AT-rich binding sites, and the lack of specific antibodies has precluded rigorous examination. To circumvent these limitations, we have generated two mouse models in which a 3XFLAG epitope tag has been inserted into the 5' end of the coding sequence of both Hoxa11 and Hoxd11 loci via Cas9/CRISPR. The alleles have been validated by sequencing, PCR genotyping, western blotting, and protein expression analyses, demonstrating proper targeting and expression. Breeding these alleles in combination produces viable and fertile Hoxa11FLAG/FLAG; Hoxd11FLAG/FLAG animals, with no overt patterning defects unlike Hoxa11/Hoxd11 mutants that are infertile and have severe kidney and limb defects. By performing CUT&RUN and CUT&Tag analyses, we have confirmed DNA binding to a known Six2 enhancer in the developing kidney. These novel alleles will allow characterization of the genome-wide binding profile of Hox11 proteins in vivo.
Previous studies of the olive ridley Lepidochelys olivacea population structure in the tropical eastern Pacific have indicated the existence of a single panmictic population ranging from Costa Rica to Mexico. This information has been used to design specific management measures to conserve primary nesting beaches in Mexico. However, little is known about olive ridleys in the Baja California Peninsula, their northernmost reproductive limit, where recent observations have shown differences in nesting female behaviour and size of hatchlings relative to other continental rookeries. We used mtDNA control region sequences from 137 turtles from five continental and four peninsular nesting sites to determine whether such differences correspond to a genetic distinction of Baja California olive ridleys or to phenotypic plasticity associated with the extreme environmental nesting conditions of this region. We found that genetic diversity in peninsular turtles was significantly lower than in continental nesting colonies. Analysis of molecular variance revealed a significant population structure (Phi ST = 0.048, P = 0.006) with the inclusion of peninsular samples. Our results: (i) suggest that the observed phenotypic variation may be associated with genetic differentiation and reproductive isolation; (ii) support the recent colonization of the eastern Pacific by Lepidochelys; (iii) reveal genetic signatures of historical expansion and colonization events; and (iv) significantly challenge the notion of a single genetic and conservation unit of olive ridleys in the eastern Pacific. We conclude that conservation measures for olive ridleys in Mexico should be revised to grant peninsular beaches special attention.
The sudden increase in information derived from the completed Mycobacterium tuberculosis (Mtb) genome sequences has revealed the need for approaches capable of converting raw genome sequence data into functional information. To date, an experimental system for studying protein-protein association in mycobacteria is not available. We have developed a simple system, termed mycobacterial protein fragment complementation (M-PFC), that is based upon the functional reconstitution of two small murine dihydrofolate reductase domains independently fused to two interacting proteins. Using M-PFC, we have successfully demonstrated dimerization of yeast GCN4, interaction between Mtb KdpD and KdpE, and association between Esat-6 and Cfp-10. We established the association between the sensor kinase, DevS, and response regulator, DevR, thereby demonstrating the potential of M-PFC to study protein associations in the mycobacterial membrane. To validate our system, we screened an Mtb library for proteins that associate with the secreted antigen Cfp-10 and consistently identified Esat-6 in our screens. Additional proteins that specifically associate with Cfp-10 include Rv0686 and Rv2151c (FtsQ), a component and substrate, respectively, of the evolutionary conserved signal recognition pathway; and Rv3596c (ClpC1), an AAA-ATPase chaperone involved in protein translocation and quality control. Our results provide empirical evidence that directly links the Mtb specialized secretion pathway with the evolutionary conserved signal recognition and SecA/SecYEG pathways, suggesting they share secretory components. We anticipate that M-PFC will be a major contributor to the systematic assembly of mycobacterial protein interaction maps that will lead to the development of better strategies for the control of tuberculosis.
Nonsynonymous single nucleotide polymorphisms (nsSNPs) alter the encoded amino acid sequence, and are thus likely to affect the function of the proteins, and represent potential disease-modifiers. There is an enormous number of nsSNPs in the human population, and the major challenge lies in distinguishing the functionally significant and potentially disease-related ones from the rest. In this study, we analyzed the genetic variations that can alter the functions and the interactions of a group of cell cycle proteins (n = 60) and the proteins interacting with them (n = 26) using computational tools. As a result, we extracted 249 nsSNPs from 77 cell cycle proteins and their interaction partners from public SNP databases. Only 31 (12.4%) of the nsSNPs were validated. The majority (64.5%) of the validated SNPs were rare (minor allele frequencies < 5%). Evolutionary conservation analysis using the SIFT tool suggested that 16.1% of the validated nsSNPs may disrupt the protein function. In addition, 58% of the validated nsSNPs were located in functional protein domains/motifs, which together with the evolutionary conservation analysis enabled us to infer possible biological consequences of the nsSNPs in our set. Our study strongly suggests the presence of naturally occurring genetic variations in the cell cycle proteins that may affect their interactions and functions with possible roles in complex human diseases, such as cancer.
By means of proviral tagging in combination with in vitro selection for invasive T-lymphoma variants, we have previously identified the murine invasion- and metastasis-inducing Tiam1 gene. Tiam1 encodes a novel protein which shares a Dbl-homology (DH) domain with GDP dissociation stimulator-(GDS) proteins that activate Rho-like GTPases. We have cloned the human TIAM1 coding sequence and studied its evolutionary conservation and expression pattern. TIAM1 is highly conserved among vertebrates. The close similarity between human TIAM1 and the mouse homologue is indicated by 88% and 95% identity of nucleotides and predicted sequences, respectively. The murine gene is highly expressed in brain and testis and at low or moderate levels in almost all other normal tissues. Interestingly, Tiam1 transcripts were found in virtually all analysed tumor cell lines of human and rodent origin including B- and T-lymphomas, neuroblastomas, melanomas and carcinomas. The evolutionary conservation as well as the broad expression pattern of Tiam1 in most normal tissues, suggests a general function in cellular signaling processes presumably by activation of a Rho-like GTPase that regulates the cytoskeletal organization.
In an experimental rat model, we recently mapped an arthritis susceptibility locus to the distal part of Chromosome 4 containing genes predicted to encode C-type lectin superfamily (CLSF) receptors. Here we report the cDNA cloning and positional arrangement of these receptor genes, which represent rat orthologues to human Mincle and DCIR and to mouse MCL and Dectin-2, as well as four novel receptors DCIR2, DCIR3, DCIR4 and DCAR1, not previously reported in other species. We furthermore report the cDNA cloning of human Dectin-2 and MCL, and of the mouse orthologues to the novel rat receptors. Similar to the killer-cell lectin-like receptors (KLR) some of these receptors exhibit structural features suggesting that they regulate leukocyte reactivity; e.g., human DCIR and rodent DCIR1 and DCIR2 carry an immunoreceptor tyrosine-based inhibitory motif (ITIM), predicting inhibitory function, and conversely, in all three species Mincle has a positively charged amino acid in the transmembrane region, suggesting activating function. Sequence comparisons show that the receptors form a discrete family, more closely related to group II CLSF receptors than to the group V KLR. Their distance to the KLR is underscored by their preservation of evolutionary conserved calcium/saccharide binding residues, present in group II and lacking in group V CLSF and their cellular expression patterns, with most of the genes preferentially expressed by professional antigen-presenting cells (dendritic cells, macrophages and B cells) and neutrophils. In all three species, the genes map together, forming an evolutionary conserved gene complex, which we call the antigen presenting lectin-like receptor complex (APLEC).
During pollen development, transcription of a large number of genes results in the appearance of distinct sets of transcripts. Similar mRNA sets are present in pollen of both mono- and dicotyledonous plant species, which indicates an evolutionary conservation of genetic programs that determine pollen gene expression. In pollen, regulation of gene expression occurs at the transcriptional and posttranscriptional level. The 5'-untranslated region (UTR) of several pollen transcripts has been shown to be important for regulation of pollen gene expression. The important regulatory role of 5'-UTR sequences and the evolutionary conservation of genetic programs in pollen led to the hypothesis that the 5'-UTRs of pollen-expressed genes share regulatory sequence elements. In an attempt to identify these pollen 5'-UTR elements, a statistical analysis was performed using 5'-UTR sequences of pollen- and sporophytic-expressed genes. The analysis revealed the presence of several pollen-specific 5'-UTR sequence elements. Assembly of the pollen 5'-UTR elements led to the identification of various consensus sequences, including those that previously have been demonstrated to play a role in the regulation of pollen gene expression. Several pollen 5'-UTR elements were found to be preferentially associated to genes from dicots, wet-type stigma plants, or plants containing bicellular pollen. Moreover, three sequence elements exhibited a preferential association to the 5'-UTR of pollen-expressed genes from Arabidopsis and Brassica napus. Functional implications of these observations are discussed.
Many peptide precursors encode more than one bioactive peptide. Recent cloning of the rat neuromedin U (NmU) precursor revealed potential proteolytic processing sites which may generate three associated peptides in addition to the NmU peptide, which is known to have potent uterine contractile effects. To assess the degree of evolutionary conservation, which often suggests conserved biological function and hence physiological importance, we have cloned and sequenced the cDNA encoding the human NmU precursor. Sequence analysis revealed a 174 amino acid human precursor containing the 25 residue NmU peptide near the C terminus of the precursor. The human message sequence was 74% homologous with that of the rat, indicating evolutionary conservation of the precursor between these two species. Four out of five of the putative proteolytic processing sites, first revealed in the rat precursor, were conserved in the human precursor, indicating a similar processing mechanism in both species. Two such processing sites flank a 33 residue peptide sequence which differed in only two amino acids compared with the rat homologue. This conservation suggests a possible biological role for this putative peptide. Northern blot analysis of human gastrointestinal tissues revealed a similar level of mRNA throughout the gastrointestinal tract. RIA using a porcine specific assay showed the highest levels of peptide in the jejunum samples.
The evolutionary conserved region of the equine homologue of the p53 gene from the donkey genome was PCR amplified and cloned. The 1380 bp fragment consisted of exons 5 to 9 and the intervening introns. The exonic and intronic DNA sequences showed a variable but high level of homology with previously published human sequences. The aminoacid sequences corresponding to the evolutionary conserved domains II, III, and V were identical to the human regions, whilst domain IV was 96% homologous.
Ca2(+)-dependent cell adhesion molecules (CAMs) are transmembrane glycoproteins structurally and functionally related in mammalian and avian species. This suggests that Ca2(+)-dependent CAMs consist of an evolutionary conserved gene family. Antibodies or cDNA probes specific either to the extracellular part or the cytoplasmic domain of uvomorulin were compared for their ability to detect corresponding molecules in Xenopus. Only antibodies directed against the evolutionary highly conserved cytoplasmic domain afforded a clear membrane staining on sections of Xenopus embryos or on cultured Xenopus epithelial cells. However, these antibodies recognized different polypeptides of 156, 140 and 128 kDa in immunoblots prepared from cell lysates of epithelial, neural, muscle and embryonic tissues. In concordance with the antibody analysis, signals in Northern hybridizations were only obtained when the cDNA probe encoding the cytoplasmic domain of uvomorulin was used. Here again, this cDNA probe revealed different mRNA species of 4.3, 4.1, 3.8 and 3.2 kb in the studied cell types. These results provide further direct evidence that the Ca2(+)-dependent CAMs are evolutionary conserved. The variety of polypeptides and transcripts observed in Xenopus indicates that several members of this gene family were detected by the use of probes specific to conserved sequences. More important, with this approach we also identified members of this gene family in the early stages of Xenopus development. Since these proteins were present in mature eggs but not in oocytes, we assume a maternal store of Ca2(+)-dependent CAM RNAs whose translation might be initiated during egg maturation.
Comparative genome analysis may provide novel insights into gene evolution and function. To investigate the von Hippel-Lindau (VHL) disease tumor suppressor gene, we sequenced the VHL gene in seven primate species. Comparative analysis was performed for human, primate, and rodent VHL genes and for a putative Caenorhabditis elegans VHL homologue identified by database analysis. The VHL gene has two translation initiation sites (at codons 1 and 54); however, the relative importance of the full-length translation product (pVHL30) and that translated from the second internal translation initiation site (pVHL19) is unclear. The N-terminal sequence of pVHL30 contains eight copies of a GXEEX acidic repeat motif in human and higher primates, but only three copies were present in the marmoset, and only one copy was present in rodent VHL genes. Evolutionary analysis suggested that the N-terminal repetitive sequence in pVHL30 was of less functional importance than those regions present in both pVHL30 and pVHL19. The VHL gene product is reported to form complexes with various proteins including elongin B, elongin C, VBP-1, fibronectin, Spl, CUL2, and HIF-1. Although most of the regions in pVHL that had been implicated in binding specific proteins demonstrated evolutionary conservation, the carboxy-terminal putative VBP-1 binding site was less well conserved, suggesting that VBP-1 binding may have less functional significance. Although an amino acid substitution (K171T) close to the pVHL elongin binding region was found in baboon, analysis of the structure of human pVHL suggested that this substitution would not interfere with pVHL/elongin C interaction. In general, there was a good correlation between the pVHL domains that demonstrated most evolutionary conservation and those that were most frequently mutated in tumors. Analysis of human/C. elegans conservation and human germline and somatic mutation patterns identified a highly conserved mutation cluster region between codons 74 and 90. However, this region is likely to be important for the structural integrity of pVHL rather than representing an additional protein binding domain.
In bacteria, synonymous codon usage can be considerably affected by base composition at neighboring sites. Such context-dependent biases may be caused by either selection against specific nucleotide motifs or context-dependent mutation biases. Here we consider the evolutionary conservation of context-dependent codon bias across 11 completely sequenced bacterial genomes. In particular, we focus on two contextual biases previously identified in Escherichia coli; the avoidance of out-of-frame stop codons and AGG motifs. By identifying homologues of E. coli genes, we also investigate the effect of gene expression level in Haemophilus influenzae and Mycoplasma genitalium. We find that while context-dependent codon biases are widespread in bacteria, few are conserved across all species considered. Avoidance of out-of-frame stop codons does not apply to all stop codons or amino acids in E. coli, does not hold for different species, does not increase with gene expression level, and is not relaxed in Mycoplasma spp., in which the canonical stop codon, TGA, is recognized as tryptophan. Avoidance of AGG motifs shows some evolutionary conservation and increases with gene expression level in E. coli, suggestive of the action of selection, but the cause of the bias differs between species. These results demonstrate that strong context-dependent forces, both selective and mutational, operate on synonymous codon usage but that these differ considerably between genomes.
Zebrafish has emerged as a valuable model for immunological studies. However, little is known about the overall picture of its immune response to infectious pathogens. Here we present the first systematic study of its immune response to Aeromonas salmonicida and Staphylococcus aureus, a Gram-negative and a Gram-positive bacteria, respectively. Genes induced upon infection were identified with suppression subtractive hybridization, with many of them encoding acute phase proteins (APPs). When compared with mammals, striking similarities and obvious differences have been observed. Both similar APPs (SAA, hepcidin and haptoglobin, etc.) and a similar system for the induction of APPs (which involves the TLRs, pro-inflammatory cytokines and C/EBPs) were identified, implying evolutionary conserved mechanisms among fish and mammals. Some novel APPs were also discovered, suggesting different immune strategies adopted by fish species. Among which, LECT2 was induced by up to 1000-fold upon infection, shedding new lights on the function of this gene. Our results constitute the first demonstration of a similar while different immune response in zebrafish and open new avenues for the investigation of evolutionary conserved and fish specific mechanisms of innate immunity.
We report the sequences of cDNA clones for two different integrin beta subunits isolated from a Xenopus laevis neurula cDNA library. mRNAs corresponding to both genes are first detected at gastrulation. We show that these two beta subunits are very highly related (98% identity in amino acid sequence) and probably arose at the time of tetraploidization of the X. laevis genome around 50 million years ago. Comparison of these sequences with those of various other vertebrate integrin beta subunit establishes that all species analyzed to date contain a highly conserved integrin beta subunit (beta 1). The interspecies homologies within this class of integrin beta subunits (82-86% identity in amino acid sequence) are much greater than those among the three different beta subunits which are known in humans (40-48% identity in amino acid sequence). Analysis of the homologies clearly indicates duplication and divergence of this multigene family more than 500 million years ago prior to the appearance of the vertebrates. We also observe cross-hybridization between cDNA probes for chicken integrin beta subunits and genomic DNAs of several invertebrate species. Despite the divergence in sequence among different integrin beta subunits, certain features of their structure are remarkably conserved.
Membrane transporters maintain cellular and organismal homeostasis by importing nutrients and exporting toxic compounds. Transporters also play a crucial role in drug response, serving as drug targets and setting drug levels. As part of a pharmacogenetics project, we screened exons and flanking intronic regions for variation in a set of 24 membrane transporter genes (96 kb; 57% coding) in 247 DNA samples from ethnically diverse populations. We identified 680 single nucleotide polymorphisms (SNPs), of which 175 were synonymous and 155 caused amino acid changes, and 29 small insertions and deletions. Amino acid diversity (pi(NS)) in transmembrane domains (TMDs) was significantly lower than in loop domains, suggesting that TMDs have special functional constraints. This difference was especially striking in the ATP-binding cassette superfamily and did not parallel evolutionary conservation: there was little variation in the TMDs, even in evolutionarily unconserved residues. We used allele frequency distribution to evaluate different scoring systems (Grantham, blosum62, SIFT, and evolutionarily conservedevolutionarily unconserved) for their ability to predict which SNPs affect function. Our underlying assumption was that alleles that are functionally deleterious will be selected against and thus under represented at high frequencies and over represented at low frequencies. We found that evolutionary conservation of orthologous sequences, as assessed by evolutionarily conservedevolutionarily unconserved and SIFT, was the best predictor of allele frequency distribution and hence of function. European Americans had an excess of high frequency alleles in comparison to African Americans, consistent with a historic bottleneck. In addition, African Americans exhibited a much higher frequency of population specific medium-frequency alleles than did European Americans.
In a recent study that identified highly evolutionary conserved sequences in three genomes of Diptera species we described an ultraconserved element found at an internal exon-intron junction of the Drosophila melanogaster homothorax (hth) gene that appeared to be involved in the control of hth pre-mRNA splicing. We also discussed a possible role of RNA secondary structure at this site in the regulation of hth pre-mRNA splicing. In this report we identify a shorter evolutionary conserved intronic element within the hth gene that is located downstream of the first element and has sequence complementarity to it. We demonstrate that intramolecular interactions between these two elements would give rise to alternative RNA secondary structures, which in turn may result in differential control of homothorax pre-mRNA splicing. We also provide additional comparative genomic data from several newly available insect genomes supporting our original conclusion that these conserved elements are important in the post-transcriptional regulation of homothorax gene expression in Diptera.