A physical map of the ribosomal DNA repeat unit of Aspergillus nidulans

The ribosomal DNA repeat unit of Aspergillus nidulans has been cloned in pBR322 and a restriction map constructed. The genes coding for the 17S, 5.8S and 25S rRNAs are found in blocks separated by a 1.7 kb spacer region, with the 5.8S RNA gene lying between the genes for the two larger RNAs. The total length of the repeat unit is 7.7 kb. The 5S rRNA is not present in the repeat unit.     

Efficient Production of Artificially Designed Gelatins with a Bacillus brevis System

Artificially designed gelatins comprising tandemly repeated 30-amino-acid peptide units derived from human αI collagen were successfully produced with a Bacillus brevis system. The DNA encoding the peptide unit was synthesized by taking into consideration the codon usage of the host cells, but no clones having a tandemly repeated gene were obtained through the above-mentioned strategy. Minirepeat genes could be selected in vivo from a mixture of every possible sequence encoding an artificial gelatin by randomly ligating the mixed sequence unit and transforming it into Escherichia coli. Larger repeat genes constructed by connecting minirepeat genes obtained by in vivo selection were also stable in the expression host cells. Gelatins derived from the eight-unit and six-unit repeat genes were extracellularly produced at the level of 0.5 g/liter and easily purified by ammonium sulfate fractionation and anion-exchange chromatography. The purified artificial gelatins had the predicted N-terminal sequences and amino acid compositions and a solgel property similar to that of the native gelatin. These results suggest that the selection of a repeat unit sequence stable in an expression host is a shortcut for the efficient production of repetitive proteins and that it can conveniently be achieved by the in vivo selection method. This study revealed the possible industrial application of artificially designed repetitive proteins.     

Diversity of O-Antigen Repeat Unit Structures Can Account for the Substantial Sequence Variation of Wzx Translocases

The most common system for synthesis of cell surface polysaccharides is the Wzx/Wzy-dependent pathway, which involves synthesis, on the cytoplasmic face of the cell membrane, of repeat units, which are then translocated to the periplasmic face by a Wzx translocase and then polymerized by Wzy to generate the polysaccharide. One such polysaccharide is O antigen, which is incorporated into lipopolysaccharide (LPS). The O antigen is extremely variable, with over 186 forms in Escherichia coli. Wzx proteins are also very diverse, but they have been thought to be specific only for the first sugar of the repeat units. However, recent studies demonstrated examples in which Wzx translocases have considerable preference for their native repeat unit, showing that specificity can extend well beyond the first sugar. These results appear to be in conflict with the early conclusions, but they involved specificity for side branch residues and could be a special case. Here we take six Wzx translocases that were critical in the earlier studies on the importance of the first sugar and assess their ability to translocate the Escherichia coli O16 and O111 repeat units. We use gene replacements to optimize maintenance of expression level and show that under these conditions the native translocases are the most effective for their native repeat unit, being, respectively, 64-fold and 4-fold more effective than the next best. We conclude that Wzx translocases are commonly adapted to their native repeat unit, which provides an explanation for the great diversity of wzx genes.     

Sequence of the cryptic satellite DNA from the plant Sinapis alba

A satellite DNA with a buoyant density equal to that of main band DNA in neutral cesium chloride (‘cryptic satellite’) can be isolated from the DNA of mustard (Sinapis alba) nuclei by Ag⁺/Cs₂SO₄ density gradient centrifugation. This satellite is cleaved into 172 bp repeat units by HinfI, AluI or HaeIII. The HinfI fragments have been further cleaved by AluI, and seven AluI subfragments have been sequenced. As a result two versions of a basic 172 HinfI repeat have been found, one (A + B) with an additional HinfI site. These two sequences (A + B and C) are the most frequent versions of the basic repeat of mustard satellite DNA. The basic 172 bp unit does not contain subrepeats or palindromic sequences. It is not similar (at a criterion of 15 common bases) with any known satellite sequence. It is not unusually highly methylated in the native state.     

The organization of sea urchin histone genes

Sucrose gradient analysis of total sea urchin DNA cleaved with theEcoRI andHind III restriction endonucleases and identification of histone coding gene sequences by hybridization with histone mRNA have elucidated the basic organization of the histone gene repeat unit. These data, plus results obtained by electrophoretic analysis of purified endonuclease-cleaved sea urchin histone DNA and hybridization with cRNA transcribed from the eucaryotic segment of constructed plasmid chimeras cloned in E. coli, show that the several DNA sequences coding for individual histone proteins are intermingled in a 7 kilobase (kb) repeat unit. Cleavage of total sea urchin DNA withEcoRI produces 2.2 and 4.8 kb fragments which are homologous with the two cloned fragments, and which are contained in a 7 kbHind III fragment. Cleavage with both enzymes reveals that the 2.2 kbEcoRI fragment contains aHind III site 0.15–0.2 kb from an end. RNA · DNA hybridization between chimeric plasmid DNA and purified individual mRNAs isolated from sea urchin embryo polyribosomes has been used to assign coding sequences to either the 2.2 or 4.8 kb region of the histone DNA repeat unit. A map of the histone genes is proposed.     

A tandem array of 5S ribosomal RNA genes in Pythium irregulare

The 5S ribosomal RNA genes of the oomycete Pythium irregulare exist in tandem arrays unlinked to the rDNA repeat unit. A clone with a 9.2-kb insert containing an array of 5S genes was identified in a lambda genomic library and was characterized by restriction mapping and partial sequencing. The array consisted of 9 apparently identical 5S genes and their spacers in tandem, followed by a diverged 5S-like sequence that is likely to be a pseudogene. This gene arrangement, although almost universal in plants and animals, is rare in fungi and protists.     

Histone genes of the sea urchin (S. purpuratus) cloned in E. coli: Order,polarity, and strandedness of the five histone-coding and spacer regions

Sea urchin (S. purpuratus) histone DNA of constructed plasmid chimeras cloned in E. coli was cleaved with the restriction endonucleases Eco RI, Hind III, Sal I, Bam I, and Hha I. The resulting fragments were ordered and isolated directly from agarose gels or cloned into other plasmids. Each fragment hybridized to one or another of the five histone mRNAs and elucidated the order of the histone genes in each of the cloned fragments. Some DNA did not hybridize to histone mRNAs and was identified as spacer DNA located between coding regions.Total sea urchin DNA was cleaved with restriction endonucleases, fractionated on agarose gels, and hybridized to histone mRNAs or histone DNA. The results revealed the order of the five histone genes in the histone gene repeat unit and demonstrate that the histone spacer DNAs have little sequence homology to other genes. Exonuclease III digestion of specific linear chimeric histone DNA plasmids followed by hybridization with mRNAs demonstrated the existence of all five histone genes on one strand of DNA and the 5′-3′ polarity of that strand. These results, in conjunction with the data of Wu et al. (1976), allow us to construct a map of coding and spacer sequences in the transcribed strand of the S. purpuratus histone gene repeat unit:

     

Comparative Mitogenomics of the Assassin Bug Genus Peirates (Hemiptera: Reduviidae: Peiratinae) Reveal Conserved Mitochondrial Genome Organization of P. atromaculatus,P. fulvescens and P. turpis

In this study, we sequenced four new mitochondrial genomes and presented comparative mitogenomic analyses of five species in the genus Peirates (Hemiptera: Reduviidae). Mitochondrial genomes of these five assassin bugs had a typical set of 37 genes and retained the ancestral gene arrangement of insects. The A+T content, AT- and GC-skews were similar to the common base composition biases of insect mtDNA. Genomic size ranges from 15,702 bp to 16,314 bp and most of the size variation was due to length and copy number of the repeat unit in the putative control region. All of the control region sequences included large tandem repeats present in two or more copies. Our result revealed similarity in mitochondrial genomes of P. atromaculatus, P. fulvescens and P. turpis, as well as the highly conserved genomic-level characteristics of these three species, e.g., the same start and stop codons of protein-coding genes, conserved secondary structure of tRNAs, identical location and length of non-coding and overlapping regions, and conservation of structural elements and tandem repeat unit in control region. Phylogenetic analyses also supported a close relationship between P. atromaculatus, P. fulvescens and P. turpis, which might be recently diverged species. The present study indicates that mitochondrial genome has important implications on phylogenetics, population genetics and speciation in the genus Peirates.     

Complete mitochondrial DNA sequence of the ark shell Scapharca broughtonii: An ultra-large metazoan mitochondrial genome

The complete mitochondrial (mt) genome of the ark shell Scapharca broughtonii was determined using long PCR and a genome walking sequencing strategy with genus-specific primers. The S. broughtonii mt genome (GenBank accession number AB729113) contained 12 protein-coding genes (the atp8 gene is missing, as in most bivalves), 2 ribosomal RNA genes, and 42 transfer tRNA genes, in a length of 46,985 nucleotides for the size of mtDNA with only one copy of the heteroplasmic tandem repeat (HTR) unit. Moreover the S. broughtonii mt genome shows size variation; these genomes ranged in size from about 47 kb to about 50 kb because of variation in the number of repeat sequences in the non-coding region. The mt-genome of S. broughtonii is, to date, the longest reported metazoan mtDNA sequence. Sequence duplication in non-coding region and the formation of HTR arrays were two of the factors responsible for the ultra-large size of this mt genome. All the tRNA genes were found within the S. broughtonii mt genome, unlike the other bivalves usually lacking one or more tRNA genes. Twelve additional specimens were used to analyze the patterns of tandem repeat arrays by PCR amplification and agarose electrophoresis. Each of the 12 specimens displayed extensive heteroplasmy and had 8–10 length variants. The motifs of the HTR arrays are about 353–362 bp and the number of repeats ranges from 1 to 11.     

Nucleotide sequence of a portion of human chromosome 9 containing a leukocyte interferon gene cluster

The nucleotide sequence of a 9937 base-pair portion of human chromosome 9, which contains two complete leukocyte interferon genes (LeIF-L and J), the complete intergenic region, and part of a third related possible pseudogene (LeIF-M), has been determined. The coding regions of the L and J genes are separated by 4363 nucleotides. The coding regions for the putative L and J interferons are 96% homologous and are each surrounded by about 3500 nucleotides of flanking sequences, which are also highly homologous. The L and J genes and their respective flanking sequences comprise a 4000 nucleotide leukocyte interferon gene repeat unit; the L gene repeat unit contains two major insertions not present in the J gene repeat unit. The J gene repeat unit is flanked by sequence features reminiscent of those found surrounding transposable elements. Both the L and J gene repeat units are embedded within sequences that are highly repeated in the human genome. Structural features identified within this portion of chromosome 9 may have been important for the generation of this interferon gene cluster.     

Genetic and Biochemical Characterizations of Enzymes Involved in Streptococcus pneumoniae Serotype 2 Capsule Synthesis Demonstrate that Cps2T (WchF) Catalyzes the Committed Step by Addition of β1-4 Rhamnose,the Second Sugar Residue in the Repeat Unit

Five genes (cps2E, cps2T, cps2F, cps2G, and cps2I) are predicted to encode the glycosyltransferases responsible for synthesis of the Streptococcus pneumoniae serotype 2 capsule repeat unit, which is polymerized to yield a branched surface structure containing glucose-glucuronic acid linked to a glucose-rhamnose-rhamnose-rhamnose backbone. Cps2E is the initiating glycosyltransferase, but experimental evidence supporting the functions of the remaining glycosyltransferases is lacking. To biochemically characterize the glycosyltransferases, the donor substrate dTDP-rhamnose was first synthesized using recombinant S. pneumoniae enzymes Cps2L, Cps2M, Cps2N, and Cps2O. In in vitro assays with each of the glycosyltransferases, only reaction mixtures containing recombinant Cps2T, dTDP-rhamnose, and the Cps2E product (undecaprenyl pyrophosphate glucose) generated a new product, which was consistent with lipid-linked glucose-rhamnose. cps2T, cps2F, and cps2I deletion mutants produced no detectable capsule, but trace amounts of capsule were detectable in Δcps2G mutants, suggesting that Cps2G adds a nonbackbone sugar. All Δcps2F, Δcps2G, and Δcps2I mutants contained different secondary suppressor mutations in cps2E, indicating that the initial mutations were lethal in the absence of reduced repeat unit synthesis. Δcps2T mutants did not contain secondary mutations affecting capsule synthesis. The requirement for secondary mutations in mutants lacking Cps2F, Cps2G, and Cps2I indicates that these activities occur downstream of the committed step in capsule synthesis and reveal that Cps2T catalyzes this step. Therefore, Cps2T is the β1-4 rhamnosyltransferase that adds the second sugar to the repeat unit and, as the committed step in type 2 repeat unit synthesis, is predicted to be an important point of capsule regulation.     

Restriction site polymorphism in rDNA repeat unit ofHordeum vulgare for some restriction endonucleases

Restriction fragment length polymorphism in rDNA repeat unit for eight restriction endonucleases was studied in two genotypes of barley (Hordeum vulgare L.). Both genotypes contained restriction sites for EcoRI, EcoRV, PvuII and Sau3239I in rDNA repeat unit and contained two different rDNA repeat unit length variants 9.7 kbp and 9.1 kbp.     

Rapid and concerted evolution of repeat units in a balbiani ring gene

The Balbiani ring (BR) genes in the midge Chironomus, a genus belonging to Diptera, code for large secretory proteins, used to construct the larval tube. The 15-23-kb long core block in each gene consists of an array of tandemly arranged approximately 200-bp long repeat units, where a single repeat unit is composed of a constant and a subrepeat region. In order to investigate the evolutionary fate of highly repetitive coding DNA, the BR1γ core block in Chironomus pallidivittatus was characterized and compared to the orthologous core block in the sibling species Chironomus tentans. We find that the 75-100 repeat units in the BR1γ core block have evolved in an unusual fashion. In all repeat units the constant regions display an expected high degree of homology between the two species, 94% at the nucleotide level. In contrast, the subrepeat regions in all repeat units have diverged concertedly, both as to length, number and sequence of the subrepeats. The observed changes in all repeat units of the core block probably have occurred after speciation of C. pallidivittatus and C. tentans. These findings demonstrate that a tandemly reiterated coding sequence can rapidly and concertedly convert into a related sequence, much in the same way as has been described for satellite DNA.     

The Diversity and Evolution of Wolbachia Ankyrin Repeat Domain Genes

Ankyrin repeat domain-encoding genes are common in the eukaryotic and viral domains of life, but they are rare in bacteria, the exception being a few obligate or facultative intracellular Proteobacteria species. Despite having a reduced genome, the arthropod strains of the alphaproteobacterium Wolbachia contain an unusually high number of ankyrin repeat domain-encoding genes ranging from 23 in wMel to 60 in wPip strain. This group of genes has attracted considerable attention for their astonishing large number as well as for the fact that ankyrin proteins are known to participate in protein-protein interactions, suggesting that they play a critical role in the molecular mechanism that determines host-Wolbachia symbiotic interactions. We present a comparative evolutionary analysis of the wMel-related ankyrin repeat domain-encoding genes present in different Drosophila-Wolbachia associations. Our results show that the ankyrin repeat domain-encoding genes change in size by expansion and contraction mediated by short directly repeated sequences. We provide examples of intra-genic recombination events and show that these genes are likely to be horizontally transferred between strains with the aid of bacteriophages. These results confirm previous findings that the Wolbachia genomes are evolutionary mosaics and illustrate the potential that these bacteria have to generate diversity in proteins potentially involved in the symbiotic interactions.     

Ribosomal RNA gene variation in diploid and tetraploid Tolmiea menziesii

Evidence from gross morphology, karyology and flavonoid chemistry suggests that Tolmiea menziesii is one of the clearest examples of autopolyploidy in natural populations. To provide additional data regarding the origin of the tetraploid cytotype of Tolmiea, both the 5S and 18S-25S ribosomal RNA genes were studied at the restriction enzyme level. Using restriction enzymes that cut once per repeat, the lengths of the 5S and 18S-25S ribosomal genes were estimated in diploids and tetraploid plants. There appear to be no consistent differences between diploids and tetraploids for the repeat length of the 18S-25S ribosomal genes. Furthermore, there is no significant repeat length heterogeneity within tetraploid plants for these genes. In addition, no differences in repeat length of the 5S genes were observed among the diploid and tetraploid plants analysed. The homogeneity observed among diploid and tetraploid plants for repeat length of the 5S and 18S-25S ribosomal genes is consistent with the hypothesis that the tetraploid cytotype is of autopolyploid origin.     

UbC gene allele frequency in Korean population and novel UbC mosaic repeat unit formation

The genomic structural organization of human UbC CDS repeat units could be representative of concerted evolution. The structure of the UbC gene and its repeat unit number frequency at scales of different human ethnic populations remain to be sufficiently determined. In this study, we performed comparative analysis of UbC CDS regions in genomes from 140 Korean individuals. We found that the UbC gene allele types 9, 8 and 7 are present in the Korean population in proportions of 97.1%, 0.4% and 2.5%, respectively. Interestingly, we discovered that the allele types 7 and 8 harbor the novel UbC gene mosaic repeat units 3??5 (combined between sequence parts derived from standard repeat units 3 and 5) and 8??9 (combined between sequence parts derived from standard repeat units 8 and 9) within their sequence structures, respectively. Our analysis showed that the novel mosaic repeat unit 3^5 lacks the highly human-specific amino acid S38, implying a functional consequence. These results suggest that the genomic organization of UbC repeat units is still undergoing dynamic structural changes due to concerted evolution through unequal crossing-over. Our results could represent valuable data for future investigations related to treating genetic diseases caused by UbC gene mutations and variations.     

Ribosomal DNA of Caenorhabditis elegans

We have characterized the organization of the genes coding for 18 S, 5·8 S and 26 S ribosomal RNAs in the nematode Caenorhabditis elegans. These ribosomal genes, present in about 55 copies per haploid genome, alternate in a repeating tandem array. The repeating unit is only 7000 base-pairs, containing a non-transcribed spacer of no more than 1000 base-pairs. Most of the repeating units have identical restriction maps, but one repeat contains a deletion of 2900 base-pairs, which eliminates all or part of the 18 S coding region. We have found no difference in the major ribosomal DNA restriction endonuclease cleavage patterns between two interbreeding strains of C. elegans, but found differences between C. elegans and the closely related Caenorhabditis briggsae.     

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.     

The U1 snRNA gene repeat from the sea urchin (Strongylocentrotus purpuratus): the 70 kilobase tandem repeat ends directly 3'' to a U1 gene.

Lambda phage clones containing multiple copies of the 1.1 kb tandemly repeated unit of the sea urchin (S. purpuratus) U1 RNA genes were isolated from a gene library. The 1.1 kb repeat unit encodes a single copy of the predominant U1 RNA expressed in oocytes and embryos prior to the blastula stage. The tandem repeat unit is about 80 kb in size and is probably present one time per haploid genome as judged by pulsed-field electrophoresis of sperm DNA digested with restriction enzymes which do not cut in the repeat unit. Two of the phage contained DNA flanking the repeat unit as well as several repeat units. The tandem repeat unit ends just 3' to the U1 coding region. There is only limited homology in the 5' flanking region with U1 snRNA genes from the sea urchin L. variegatus.