ZIPK: a unique case of murine-specific divergence of a conserved vertebrate gene

Zipper interacting protein kinase (ZIPK, also known as death-associated protein kinase 3 [DAPK3]) is a Ser/Thr kinase that functions in programmed cell death. Since its identification eight years ago, contradictory findings regarding its intracellular localization and molecular mode of action have been reported, which may be attributed to unpredicted differences among the human and rodent orthologs. By aligning the sequences of all available ZIPK orthologs, from fish to human, we discovered that rat and mouse sequences are more diverged from the human ortholog relative to other, more distant, vertebrates. To test experimentally the outcome of this sequence divergence, we compared rat ZIPK to human ZIPK in the same cellular settings. We found that while ectopically expressed human ZIPK localized to the cytoplasm and induced membrane blebbing, rat ZIPK localized exclusively within nuclei, mainly to promyelocytic leukemia oncogenic bodies, and induced significantly lower levels of membrane blebbing. Among the unique murine (rat and mouse) sequence features, we found that a highly conserved phosphorylation site, previously shown to have an effect on the cellular localization of human ZIPK, is absent in murines but not in earlier diverging organisms. Recreating this phosphorylation site in rat ZIPK led to a significant reduction in its promyelocytic leukemia oncogenic body localization, yet did not confer full cytoplasmic localization. Additionally, we found that while rat ZIPK interacts with PAR-4 (also known as PAWR) very efficiently, human ZIPK fails to do so. This interaction has clear functional implications, as coexpression of PAR-4 with rat ZIPK caused nuclear to cytoplasm translocation and induced strong membrane blebbing, thus providing the murine protein a possible adaptive mechanism to compensate for its sequence divergence. We have also cloned zebrafish ZIPK and found that, like the human and unlike the murine orthologs, it localizes to the cytoplasm, and fails to bind the highly conserved PAR-4 protein. This further supports the hypothesis that murine ZIPK underwent specific divergence from a conserved consensus. In conclusion, we present a case of species-specific divergence occurring in a specific branch of the evolutionary tree, accompanied by the acquisition of a unique protein–protein interaction that enables conservation of cellular function.     

The MHC class II-associated chicken invariant chain shares functional properties with its mammalian homologs

The nucleotide sequence of chicken invariant chain (Ii) was determined, and the amino acid sequence similarity with human Ii is 61%. Certain regions important for the biological function of human Ii are highly conserved between chicken and mammals. The cytoplasmic tail of chicken Ii fused to the plasma membrane reporter molecule neuraminidase relocated the protein to endosomes. Moreover, like the mammalian orthologs, the cytoplasmic tail was found to contain two independent leucine-based endosomal sorting signals. Chicken Ii was found to interact with human Ii and crosslinking studies also indicate that chicken Ii assembles as a trimer. The chicken Ii can furthermore bind the human MHC class II (HLA-DR1). Many of the functional properties between the chicken Ii and its mammalian orthologs are thus maintained in spite of their sequence differences.     

Cloning, Pharmacology, and Tissue Distribution of G-Protein-Coupled Receptor GPR105 (KIAA0001) Rodent Orthologs

It has recently been shown that UDP-glucose is a potent agonist of the orphan G-protein-coupled receptor (GPCR) KIAA0001. Here we report cloning and analysis of the rat and mouse orthologs of this receptor. In accordance with GPCR nomenclature, we have renamed the cDNA clone, KIAA0001, and its orthologs GPR105 to reflect their functionality as G-protein-coupled receptors. The rat and mouse orthologs show 80% and 83% amino acid identity, respectively, to the human GPR105 protein. We demonstrate by genomic Southern blot analysis that there are no genes in the mouse or rat genomes with higher sequence similarity. Chromosomal mapping shows that the mouse and human genes are located on syntenic regions of chromosome 3. Further analyses of the rat and mouse GPR105 proteins show that they are activated by the same agonists as the human receptor, responding to UDP-glucose and closely related molecules with similar affinities. The mouse and rat receptors are widely expressed, as is the human receptor. Thus we conclude that we have identified the rat and mouse orthologs of the human gene GPR105.     

The nucleotide sequence of the cDNA coding for the human dihydrofolic acid reductase

The nucleotide sequence of the human dihydrofolic acid reductase (DHFR) reading frame has been derived from the analysis of human DHFR cDNA. This sequence and the corresponding amino acid sequence have been compared with those available for the enzyme and its coding segment from other organisms. There is an 89% nucleotide sequence homology between the human DHFR reading frame and the mouse coding sequence. Furthermore, amino acid-sequence homologies of 74%, 81% and 89% has been found between human DHFR and chicken, bovine and mouse DHFR, respectively.     

Immunoglobulin switch region-like sequences in Drosophila melanogaster.

We found immunoglobulin switch (S) region-like sequences in DNAs of wide variety of organisms including sea urchin, yeast and Drosophila that do not produce immunoglobulins. DNA fragments carrying Smu-like sequences were cloned from Drosophila and the nucleotide sequence of a clone is almost identical to that of the mouse Smu region. Restriction fragments of Drosophila Smu-like sequences and their flanking regions seem to vary among Drosophila species. Possible evolutionary significance of the Smu-like sequence in invertebrates was discussed.     

A Model of Evolutionary Base Substitutions and Its Application with Special Reference to Rapid Change of Pseudogenes

A model of evolutionary base substitutions that can incorporate different substitutional rates between the four bases and that takes into account unequal composition of bases in DNA sequences is proposed. Using this model, we derived formulae that enable us to estimate the evolutionary distances in terms of the number of nucleotide substitutions through comparative studies of nucleotide sequences. In order to check the validity of various formulae, Monte Carlo experiments were performed. These formulae were applied to analyze data on DNA sequences from diverse organisms. Particular attention was paid to problems concerning a globin pseudogene in the mouse and the time of its origin through duplication. We obtained a result suggesting that the evolutionary rates of substitution in the first and second codon positions of the pseudogene were roughly 10 times faster than those in the normal globin genes; whereas, the rate in the third position remained almost unchanged. Application of our formulae to histone genes H2B and H3 of the sea urchin showed that, in each of these genes, the rate in the third codon position is tremendously higher than that in the second position. All of these observations can easily and consistently be interpreted by the neutral theory of molecular evolution.     

Evolutionary conservation and selection of human disease gene orthologs in the rat and mouse genomes

Background

Model organisms have contributed substantially to our understanding of the etiology of human disease as well as having assisted with the development of new treatment modalities. The availability of the human, mouse and, most recently, the rat genome sequences now permit the comprehensive investigation of the rodent orthologs of genes associated with human disease. Here, we investigate whether human disease genes differ significantly from their rodent orthologs with respect to their overall levels of conservation and their rates of evolutionary change.

Results

Human disease genes are unevenly distributed among human chromosomes and are highly represented (99.5%) among human-rodent ortholog sets. Differences are revealed in evolutionary conservation and selection between different categories of human disease genes. Although selection appears not to have greatly discriminated between disease and non-disease genes, synonymous substitution rates are significantly higher for disease genes. In neurological and malformation syndrome disease systems, associated genes have evolved slowly whereas genes of the immune, hematological and pulmonary disease systems have changed more rapidly. Amino-acid substitutions associated with human inherited disease occur at sites that are more highly conserved than the average; nevertheless, 15 substituting amino acids associated with human disease were identified as wild-type amino acids in the rat. Rodent orthologs of human trinucleotide repeat-expansion disease genes were found to contain substantially fewer of such repeats. Six human genes that share the same characteristics as triplet repeat-expansion disease-associated genes were identified; although four of these genes are expressed in the brain, none is currently known to be associated with disease.

Conclusions

Most human disease genes have been retained in rodent genomes. Synonymous nucleotide substitutions occur at a higher rate in disease genes, a finding that may reflect increased mutation rates in the chromosomal regions in which disease genes are found. Rodent orthologs associated with neurological function exhibit the greatest evolutionary conservation; this suggests that rodent models of human neurological disease are likely to most faithfully represent human disease processes. However, with regard to neurological triplet repeat expansion-associated human disease genes, the contraction, relative to human, of rodent trinucleotide repeats suggests that rodent loci may not achieve a 'critical repeat threshold' necessary to undergo spontaneous pathological repeat expansions. The identification of six genes in this study that have multiple characteristics associated with repeat expansion-disease genes raises the possibility that not all human loci capable of facilitating neurological disease by repeat expansion have as yet been identified.     

Sequence analysis of 193.4 and 83.9 kbp of mouse and chicken genomic DNAs containing the entire Prkdc (DNA-PKcs) gene

The catalytic subunit of DNA-dependent protein kinase plays critical roles in nonhomologous end joining in repair of DNA double-strand breaks and V(D)J recombination. In addition to the SCID phenotype, it has been suggested that the molecule contributes to the polymorphic variations in radiosensitivity and susceptibility to cancer in mouse strains. Here we show the nucleotide sequence of approximately 193-kbp and 84-kbp genomic regions encoding the entire Prkdc gene (also known as DNA-PKcs) in the mouse and chicken, respectively. A large retroposon was found in intron 51 in the mouse but not in the human or chicken. Comparative analyses of the genome strongly suggested that the region contains only two genes for Prkdc and Mcm4; however, several conserved sequences and cis elements were also predicted.     

Negative correlation of G+C content at silent substitution sites between orthologous human and mouse protein-coding sequences.

We conducted a genome-wide analysis of variations in guanine plus cytosine (G+C) content at the third codon position at silent substitution sites of orthologous human and mouse protein-coding nucleotide sequences. Alignments of 3776 human protein-coding DNA sequences with mouse orthologs having >50 synonymous codons were analyzed, and nucleotide substitutions were counted by comparing sequences in the alignments extracted from gap-free regions. The G+C content at silent sites in these pairs of genes showed a strong negative correlation (r = -0.93). Some gene pairs showed significant differences in G+C content at the third codon position at silent substitution sites. For example, human thymine-DNA glycosylase was A+T-rich at the silent substitution sites, while the orthologous mouse sequence was G+C-rich at the corresponding sites. In contrast, human matrix metalloproteinase 23B was G+C-rich at silent substitution sites, while the mouse ortholog was A+T-rich. We discuss possible implications of this significant negative correlation of G+C content at silent sites.     

Functional and evolutionary analyses on expressed intronless genes in the mouse genome

Using computational approaches we have identified 2017 expressed intronless genes in the mouse genome. Evolutionary analysis reveals that 56 intronless genes are conserved among the three domains of life--bacteria, archea and eukaryotes. These highly conserved intronless genes were found to be involved in essential housekeeping functions. About 80% of expressed mouse intronless genes have orthologs in eukaryotic genomes only, and thus are specific to eukaryotic organisms. 608 of these genes have intronless human orthologs and 302 of these orthologs have a match in OMIM database. Investigation into these mouse genes will be important in generating mouse models for understanding human diseases.     

Evolution of human immunoglobulin kappa J region genes

Immunoglobulin kappa chain variable region genes are assembled from two discontinuous DNA segments, a V and a J gene. The J region genes, in addition to encoding amino acid positions 96-108 of the kappa polypeptide chain, also provide sequences required for both DNA and RNA splicing reactions. For purposes of evolutionary comparison and to establish the complexity of the kappa J region locus in man, we have determined an approximately 3000 basepair nucleotide sequence in a cloned human DNA fragment that encodes the germline distinct J region segments. Significant blocks of homology have been tightly maintained between this region and an analogous segment of the mouse genome. In particular, the short sequences, GGTTTTTGT and CACTGTG, thought to be involved in V-J recombination, are the most highly conserved regions (97% homology). In addition, from heteroduplex data and computer analysis of the nucleotide sequences, it is clear that the mouse J3 sequence, a pseudogene, is not present in the human cluster. This can be explained by a duplication event in the mouse J region gene cluster that may have been the result of unequal crossing over between homologous chromosomes.