Lineage-Specific Homogenization of the Polyubiquitin Gene Among Human and Great Apes

Ubiquitin is a highly conserved protein, and is encoded by a multigene family among eukaryote species. The polyubiquitin genes, UbB and UbC, comprise tandem multiple ubiquitin coding units without a spacer region or intron. We determined nucleotide sequences for the UbB and UbC of human, chimpanzee, gorilla, and orangutan. The ubiquitin repeat number of UbB was constant (3) in human and great apes, while that of UbC varied: 6 to 11 for human, 10 to 12 for chimpanzee, 8 for gorilla, and 10 for orangutan. The heterogeneity of the repeat number within closely related hominoid species suggests that a lineage-specific unequal crossover and/or gene duplication occurred. A marked homogenization of UbC occurred in gorilla with a low level of synonymous difference (p_s). The homogenization of UbC also occurred in chimpanzee and less strikingly in human. The first and last ubiquitin coding units of UbC were clustered independently between species, and less affected by homogenization during the hominoid evolution. Therefore, the homogenization of ubiquitin coding units is likely due to an unequal crossing-over inside the ubiquitin units. The lineage-specific homogenization of UbC among closely related species suggests that concerted evolution has a key role in the short-term evolution of UbC.     

Phylogenetic analysis of the swine leukocyte antigen — 2 gene for Korean native pigs

The objective of this study was to investigate genetic relationships of the SLA-2 gene, to characterize SLA-2 alleles, and to provide basic genetic information of Korean pigs. The swine leukocyte antigen - 2 (SLA-2) gene in the MHC classical region was cloned with spleen tissues from Korean native pigs selected from the main land (KNP) and Jeju Island (KJP). Primer sequences based on swine cDNA (GenBank accession numbers AF464049 and AF464005) were used to amplify the entire SLA-2 gene, and the amplification product including both 3′ and 5′ UTRs was sized 1,520 bp. A BAC clone was selected from miniature pigs and sequenced for the genomic region of the SLA-2 gene showing that 4,585 bp in total length consisted of exons (1,087 bp) and introns (3,498 bp). A sequence analysis confirmed 58 SNPs in coding regions, which revealed higher numbers of SNPs in KNP than other pig breeds, implicating more genetic variability in Korean pigs. Approximately 82% of the SLA-2 SNPs were located in the highly polymorphic exons 2 and 3. Newly identified sequences of the SLA-2 gene for KNP and KJP were submitted into the IPD-MHC database with new nomenclatures (SLA-2*1501, SLA-2*1601, and SLA-2*w08hy01 allele), while the representative sequences of KNP and KJP were submitted into GenBank with accession numbers (DQ992495, DQ992496, and DQ992501), respectively. The identified KNP allele (SLA-2*1501) clustered with previously defined alleles for Korean pigs (SLA-2*kn02 and SLA-2*jh01), but SLA-2*1601 (KNP) and SLA-2*w08hy01 (KJP) alleles showed no significant genetic relationships with any other allele. A sequence comparison revealed that KNP has departed from KJP both genetically and phenotypically. The results of SLA-2 SNP in KNP and KJP reported here will serve as the SLA-2 reference for Korean pigs.     

Cloning and sequencing of the gene for a 120-kDa immunodominant protein of Ehrlichia chaffeensis

Ehrlichia chaffeensis is the tick-borne, obligately intracellular bacterium that causes human monocytic ehrlichiosis. A 120-kDa protein is one of the immunodominant proteins of E. chaffeensis that stimulates production of specific antibodies in infected humans. A genomic library of E. chaffeensis was constructed in a λZAP II phage vector, and a clone expressing the 120-kDa protein of E. chaffeensis was identified using canine anti-E. chaffeensis serum. DNA sequence analysis of the cloned 120-kDa protein gene of E. chaffeensis identified a 1884-bp open reading frame with an ehrlichial promoter. Five identical 240-bp tandem repeat units were identified in the 120-kDa protein gene of E. chaffeensis, comprising 60% of the entire gene. Aside from the first repeat unit, all the other repeat units are identical. In the first repeat unit there are four nucleotides that are different from the other repeats. Hydropathy analysis of the deduced amino-acid sequence demonstrated that the repeat domain contains highly hydrophilic segments. The 120-kDa protein should be evaluated for a role in stimulating protective immunity.     

Allelic variation of the dopamine receptor D4 gene polymorphic region in gibbons

We examined the tandem repeat sequence of the dopamine receptor D4 (DRD4) gene in 73 individuals derived from 8 species of gibbons (genusHylobates) in an attempt to assess the variability of this gene in gibbon species.H. syndactylus (subgenusSymphalangus) andH. concolor (subgenusNomascus), which were inferred to have diverged at an early time within the family Hylobatidae, shared only long repeat (7–8) alleles. On the other hand, DRD4 was highly polymorphic in gibbons of the subgenusHylobates, with 4-, 5-, 6-, 7-, and 8-repeat alleles being recognized. In this subgenus, 4- and 5-repeat alleles were found in the species distributed mainly in the southern islands such as Sumatra, Java, and Borneo but not in the species inhabiting the Asian continent. Sequence analysis indicated that the repeat structure of the gibbon DRD4 gene was quite complex but most of the 48-bp units could be classified into several groups across the species based on sequence similarities. However, the sequence of the 7-repeat allele ofH. muelleri was unique, since the repeat units had low similarities to other units of gibbons.     

Whole-genome association study for the roan coat color in an intercrossed pig population between Landrace and Korean native pig

The roan coat color is characterized by white hairs intermingled with colored hairs. Candidate genes based on comparative phenotypes in horses and cattle involve the KIT and KIT ligand (MGF) genes. Here, we report the result of the whole genome scanning to detect genomic regions responsible for the roan coat color, using a three-generation pedigree of 62 pigs in an intercross between Landrace and Korean native pig. These pigs were genotyped using the PorcineSNP 60 BeadChip (Illumina, USA). The whole genome scan indicated that three genomic regions, 35～36 Mb, 38～39 Mb, and 58～59 Mb on SSC8, were commonly and highly associated/linked with the roan phenotype in the case/control, sib-pair, and linkage test, respectively. The porcine KIT was selected as a candidate gene, because it is located in one of the three significant regions and its function is related to coat color formation. SNPs and Indels within coding sequence (CDS), promoter, and 3′-UTR of KIT were surveyed. Twenty-two SNPs in the CDS reported previously, as well as nine variations in promoter (2 SNPs) and 3′-UTR (5 SNPs and 2 Indels) were detected. Although no causative mutations were identified, these results will help to elucidate the genetic mechanisms involved in the expression of the roan phenotype and will aid in identifying key mutations responsible for the roan phenotype in further studies.     

Concerted Evolution of the Tandemly Repeated Genes Encoding Primate U2 Small Nuclear RNA (the RNU2 Locus) Does Not Prevent Rapid Diversification of the (CT)n · (GA)n Microsatellite Embedded within the U2 Repeat Unit

The RNU2 locus encoding human U2 small nuclear RNA (snRNA) is organized as a nearly perfect tandem array containing 5 to 22 copies of a 5.8-kb repeat unit. Just downstream of the U2 snRNA gene in each 5.8-kb repeat unit lies a large (CT)_n · (GA)_n dinucleotide repeat (n ≈ 70). This form of genomic organization, in which one repeat is embedded within another, provides an unusual opportunity to study the balance of forces maintaining the homogeneity of both kinds of repeats. Using a combination of field inversion gel electrophoresis and polymerase chain reaction, we have been able to study the CT microsatellites within individual U2 tandem arrays. We find that the CT microsatellites within an RNU2 allele exhibit significant length polymorphism, despite the remarkable homogeneity of the surrounding U2 repeat units. Length polymorphism is due primarily to loss or gain of CT dinucleotide repeats, but other types of deletions, insertions, and substitutions are also frequent. Polymorphism is greatly reduced in regions where pure (CT)_n tracts are interrupted by occasional G residues, suggesting that irregularities stabilize both the length and the sequence of the dinucleotide repeat. We further show that the RNU2 loci of other catarrhine primates (gorilla, chimpanzee, orangutan, and baboon) contain orthologous CT microsatellites; these also exhibit length polymorphism, but are highly divergent from each other. Thus, although the CT microsatellite is evolving far more rapidly than the rest of the U2 repeat unit, it has persisted through multiple speciation events spanning >35 Myr. The persistence of the CT microsatellite, despite polymorphism and rapid evolution, suggests that it might play a functional role in concerted evolution of the RNU2 loci, perhaps as an initiation site for recombination and/or gene conversion.     

An efficient and rapid method for gene cloning from eukaryotic genomic DNA using overlap-PCR: With an example of cattle Ghrelin gene

In this study, overlap-PCR, an efficient and rapid method, was used to clone cattle Ghrelin gene CDS (coding sequence) from genomic DNA. The procedure included seven primers and three-step PCRs. Cattle Ghrelin gene consists of four exons and the CDS contains 351 bps. In the first step three PCRs were performed to generate extended exon1, exon2, and exon3 that contained overlapped nucleotides and were used as the templates for second ligation PCR. Secondly, exon1 and exon2 were spliced together. And it was same with exon3 and exon4. Lastly, the four exons were linked together with outermost primers and the templates from the second step. Comparison analysis on the obtained CDS of Ghrelin gene and cDNA by RT-PCR showed that the two sequences were same. As an efficient and rapid method, overlap-PCR is feasible and acceptable for gene cloning from genomic DNA.     

Mosaicism for FMR1 gene full mutation and intermediate allele in a female foetus: A postzygotic retraction event

Fragile X syndrome is caused by the expansion of an unstable CGG repeat in the 5′UTR of FMR1 gene. The occurrence of mosaicism is not uncommon, especially in male patients, whereas in females it is not so often reported. Here we report a female foetus that was subject to prenatal diagnosis, because of her mother being a premutation carrier. The foetus was identified as being a mosaic for an intermediate allele and a full mutation of FMR1 gene, in the presence of a normal allele. The mosaic status was confirmed in three different tissues of the foetus – amniotic fluid, skin biopsy and blood – the last two obtained after pregnancy termination. Karyotype analysis and X-chromosome STR markers analysis do not support the mosaicism as inheritance of both maternal alleles. Oligonucleotide array-CGH excluded an imbalance that could contain the primer binding site with a different repeat size. The obtained results give compelling evidence for a postzygotic expansion mechanism where the foetus mosaic pattern originated from expansion of the mother's premutation into a full mutation and consequent regression to an intermediate allele in a proportion of cells. These events occurred in early embryogenesis before the commitment of cells into the different tissues, as the three tested tissues of the foetus have the same mosaic pattern. The couple has a son with Fragile X mental retardation syndrome and choose to terminate this pregnancy after genetic counselling.     

The Balbiani ring 2 gene in Chironomus tentans is built from two types of tandemly arranged major repeat units with a common evolutionary origin

The internal structure of the 37 kb long Balbiani ring 2 (BR 2) gene in Chironomus tentans has been studied by analysis of a collection of cloned cDNA sequences and in genomic Southern blot analysis with the cDNA sequences used as probes. The BR 2 gene contains two types of tandemly arranged major repeat units ˜200 bp long, represented in our study by the pCt 7 and the pCt 63 cDNA inserts. The pCt 7 major repeat units are arranged in one or possibly a few blocks and cover ˜10 kb of the gene; the pCt 63 units form one uninterrupted block, 22 kb in length. Genomic Southern blot hybridizations revealed a number of sequence variants of the pCt 7 major repeat unit. In contrast, the ˜100 copies of the pCt 63 major repeat unit seem to be almost identical. The pCt 7 major repeat unit, 180 bp in length, is organized in the same way as the previously described 215 bp long pCt 63 major repeat, i.e., it contains a repetitive and a non-repetitive part. Moreover, the two major repeat units show a high degree of sequence homology, indicating that the pCt 7 and pCt 63 sequence blocks within the Br 2 gene have evolved through stepwise amplification from a common ancestral sequence.     

Gene organization of human NOTCH4 and (CTG)n polymorphism in this human counterpart gene of mouse proto-oncogene Int3

The cDNA and genomic clones for the human counterpart of the mouse mammary tumor gene Int3 were isolated and sequenced. We designated this human major histocompatibility complex (MHC) class III gene as NOTCH4, since very recently, by sequencing cDNA clones, the complete form of the mouse proto-oncogene Int3 has been clarified and named Notch4. The present human NOTCH4 sequence is the first example of the genomic sequence for the extracellular portion of the mammalian Notch4, and by comparing it with the mouse Notch4 cDNA sequence, the exon/intron organization was clarified. The comparison of the predicted amino acid sequence of human NOTCH4 with those of other Notch homologues of a wide range of species revealed four subfamilies for mammalian Notch. In the protein coding region of human NOTCH4, we found (CTG)_n repeats showing a variable number tandem repeat (VNTR) polymorphism for different human leukocyte antigen (HLA) haplotypes. Ten genes mapped on 6p21.3, including NOTCH4, were found to have counterparts structurally and functionally similar to those mostly mapped on 9q33-q34, indicating segmental chromosome duplication during the course of evolution. Similarity of genes on chromosomes 1, 6, 9 and 19 was also discussed.     

The chAB4 and NF1-related long-range multisequence DNA families are contiguous in the centromeric heterochromatin of several human chromosomes

We have investigated the large-scale organization of the human chAB4-related long-range multisequence family, a low copy-number repetitive DNA located in the pericentromeric heterochromatin of several human chromosomes. Analysis of genomic clones revealed large-scale (~100 kb or more) sequence conservation in the region flanking the prototype chAB4 element. We demonstrated that this low copy-number family is connected to another long-range repeat, the NF1-related (ΨNF1) multisequence. The two DNA types are joined by an ~2 kb-long tandem repeat of a 48-bp satellite. Although the chAB4- and NF1-like sequences were known to have essentially the same chromosomal localization, their close association is reported here for the first time. It indicates that they are not two independent long-range DNA families, but are parts of a single element spanning ~200 kb or more. This view is consistent both with their similar chromosomal localizations and the high levels of sequence conservation among copies found on different chromosomes. We suggest that the master copy of the linked chAB4–ΨNF1 DNA segment appeared first on the ancestor of human chromosome 17.     

Evolutionary history of x-tox genes in three lepidopteran species: Origin,evolution of primary and secondary structure and alternative splicing,generating a repertoire of immune-related proteins

The proteins of the X-tox family have imperfectly conserved tandem repeats of several defensin-like motifs known as cysteine-stabilized αβ (CS-αβ) motifs. These immune-related proteins are inducible and expressed principally in hemocytes, but they have lost the antimicrobial properties of the ancestral defensins from which they evolved. We compared x-tox gene structure and expression in three lepidopteran species (Spodoptera frugiperda, Helicoverpa armigera and Bombyx mori). Synteny and phylogenetic analyses showed that the x-tox exons encoding CS-αβ motifs were phylogenetically closely related to defensin genes mapping to chromosomal positions close to the x-tox genes. We were able to define two groups of paralogous x-tox exons (three in Noctuids) that each followed the expected species tree. These results suggest that the ancestor of the three species already possessed an x-tox gene with at least two proto-domains, and an additional duplication/fusion should have occurred in the ancestor of the two noctuid species. An expansion of the number of exons subsequently occurred in each lineage. Alternatively, the proto x-tox gene possessed more copy and each group of x-tox domains might undergo concerted evolution through gene conversion. Accelerated protein evolution was detected in x-tox domains when compared to related defensins, concomitantly to multiplication of exons and/or the possible activation of concerted evolution. The x-tox genes of the three species have similar structural organizations, with repeat motifs composed of CS-αβ-encoding exons flanked by introns in phase 1. Diverse mechanisms underlie this organization: (i) the acquisition of new repeat motifs, (ii) the duplication of preexisting repeat motifs and (iii) the duplication of modules. A comparison of gDNA and cDNA structures showed that alternative splicing results in the production of multiple X-tox protein isoforms from the x-tox genes. Differences in the number and sequence of CS-αβ motifs in these isoforms were found between species, but also between individuals of the same species. Thus, our analysis of the genetic organization and expression of x-tox genes in three lepidopteran species suggests a rapid evolution of the organization of these genes.     

Heterogeneity of pumpkin ribosomal DNA

The ribosomal DNA (rDNA) of Cucurbita pepo L. has been found to consist of tandemly arrayed repeat units, most of which are 10 kilobases in length. Thirty-six repeat units, cloned into the HindIII site of pACYC 177, fall into seven classes which differ from each other in length and/or nucleotide sequence. Most of the heterogeneity occurs in noncoding portions of the repeat unit although there is some nucleotide sequence variation in the coding portion as well. Heterogeneity of base modification was observed in genomic rDNA of which two examples are: (a) all of the repeat units have three BamHI sites, one of which is unavailable for restriction in about half of the units and (b) all of the CCGG sites except one are methylated at the internal cytidine in many of the units; a second site is unmethylated in some of the units and in a very few units a third site remains unmethylated.     

A Concertedly Evolving Region in Chironomus, Unique Within the Telomere

Chromosome terminal, complex repeats in the dipteran Chironomus pallidivittatus show rapid concerted evolution during which there is remarkably efficient homogenization of the repeat units within and between chromosome ends. It has been shown previously that gene conversion is likely to be an important component during these changes. The sequence evolution could be a result of different processes—exchanges between repeats in the tandem array as well as information transfer between units in different chromosomes—and is therefore difficult to analyze in detail. In this study the concerted evolution of a region present only once per chromosome, at the junction between the telomeric complex repeats and the subtelomeric DNA was therefore investigated in the two sibling species C. pallidivittatus and C. tentans. Material from individual microdissected chromosome ends was used, as well as clones from bulk genomic DNA. On the telomeric side of the border pronounced species-specific sequence differences were observed, the patterns being similar for clones of different origin within each species. Mutations had been transmitted efficiently between chromosomes also when adjoining, more distally localized DNA showed great differences in sequence, suggesting that gene conversion had taken place. The evolving telomeric region bordered proximally to subtelomeric DNA with high evolutionary constancy. More proximally localized, subtelomeric DNA evolved more rapidly and showed heterogeneity between species and chromosomes. Received: 24 September 1997 / Accepted: 24 November 1997     

Identification, Genomic Organization, and Analysis of the Group III Capsular Polysaccharide Genes kpsD, kpsM, kpsT, and kpsE from an Extraintestinal Isolate of Escherichia coli (CP9, O4/K54/H5)

Group III capsular polysaccharides (e.g., K54) of extraintestinal isolates of Escherichia coli, similar to group II capsules (e.g., K1), are important virulence traits that confer resistance to selected host defense components in vitro and potentiate systemic infection in vivo. The genomic organization of group II capsule gene clusters has been established as a serotype-specific region 2 flanked by regions 1 and 3, which contain transport genes that are highly homologous between serotypes. In contrast, the organization of group III capsule gene clusters is not well understood. However, they are defined in part by an absence of genes with significant nucleotide homology to group II capsule transport genes in regions 1 and 3. Evaluation of isogenic, TnphoA-generated, group III capsule-minus derivatives of a clinical blood isolate (CP9, O4/K54/H5) has led to the identification of homologs of the group II capsule transport genes kpsDMTE. These genes and their surrounding regions were sequenced and analyzed. The genomic organization of these genes is distinctly different from that of their group II counterparts. Although kps_K54DMTE are significantly divergent from their group II homologs at both the DNA and protein levels phoA fusions and computer-assisted analyses suggest that their structures and functions are similar. The putative proteins Kps_K54M and Kps_K54T appear to be the integral membrane component and the peripheral ATP-binding component of the ABC-2 transporter family, respectively. The putative Kps_K54E possesses features similar to those of the membrane fusion protein family that facilitates the passage of large molecules across the periplasm. At one boundary of the capsule gene cluster, a truncated kpsM (kpsM_truncated) and its 5′ noncoding regulatory sequence were identified. In contrast to the complete kps_K54M, this region was highly homologous to the group II kpsM. Fifty-three base pairs 3′ from the end of kpsM_truncated was a sequence 75% homologous to the 39-bp inverted repeat in the IS110 insertion element from Streptomyces coelicolor. Southern analysis established that two copies of this element are present in CP9. These findings are consistent with the hypothesis that CP9 previously possessed group II capsule genes and acquired group III capsule genes via IS110-mediated horizontal transfer.     

Sequence rearrangements between mitochondrial DNAs of Torulopsis glabrata and Kloeckera africana identified by hybridization with six polypeptide encoding regions from Saccharomyces cerevisiae mitochondrial DNA

Sequences hybridizing to six mitochondrial DNA encoded polypeptide genes of Saccharomyces cerevisiae have been mapped in the 18·9 and 27·1 kbp² circular mitochondrial DNAs from Torulopsis glabrata and Kloeckera africana. With the possible exception of cytochrome oxidase subunit 1 and ATPase subunit 6 genes, no two hybridizable sequences share the same order in the two mtDNAs nor is there any topographical similarity to S. cerevisiae mtDNA apart from the grouping mentioned above. Because sequence rearrangements are prevalent in yeast mitochondrial DNAs we infer that order is not critical for mitochondrial gene expression and that prokaryotic-like operons do not exist. In contrast to S. cerevisiae, the cytochrome b region in T. glabrata and K. africana is confined to 1·46 or 1·58 kbp, respectively, which suggests that intervening sequences in this gene are either small or absent. On the other hand, hybridizable sequences to a 5·2 kbp portion of the S. cerevisiae cytochrome oxidase subunit 1 gene, retaining exons 3 to 7 or 8, span 3 to 4 kbp in the two mtDNAs. In addition an 0·8 to 0·9 kbp intervening sequence is present in each case, which does not hybridize to either exon or intron regions of the S. cerevisiae probe. These results imply that the cytochrome oxidase subunit 1 gene in both mtDNAs has a mosaic organization of coding and noncoding sequences.