Single Repeat Unit Research Articles

Inactivation of the flax rust resistance gene M associated with loss of a repeated unit within the leucine-rich repeat coding region.

The M rust resistance gene from flax was cloned after two separate approaches, an analysis of spontaneous M mutants with an L6 gene-derived DNA probe and tagging with the maize transposon Activator, independently identified the same gene. The gene encodes a protein of the nucleotide binding site leucine-rich repeat class and is related (86% nucleotide identity) to the unlinked L6 rust resistance gene. In contrast to the L locus, which contains a single gene with multiple alleles, approximately 15 related genes occur at the complex M locus, with only one encoding the M resistance specificity. The M protein contains two direct repeats of 147 and 149 amino acids in the C-terminal part of the leucine-rich region. Three mutant alleles of M encoding a product containing a single repeat unit of 154 amino acids were isolated. The mutant DNA sequences probably occurred by unequal intragenic exchange in the coding region of the repeats. The recombinant alleles lost M resistance and gained no detectable new resistance specificity.

Microsatellite and chromosome evolution of parthenogenetic sitobion aphids in Australia.

Single-locus microsatellite variation correlated perfectly with chromosome number in Sitobion miscanthi aphids. The microsatellites were highly heterozygous, with up to 10 alleles per locus in this species. Despite this considerable allelic variation, only seven different S. miscanthi genotypes were discovered in 555 individuals collected from a wide range of locations, hosts and sampling periods. Relatedness between genotypes suggests only two successful colonizations of Australia. There was no evidence for genetic recombination in 555 S. miscanthi so the occurrence of recent sexual reproduction must be near zero. Thus diversification is by mutation and chromosomal rearrangement alone. Since the aphids showed no sexual recombination, microsatellites can mutate without meiosis. Five of seven microsatellite differences were a single repeat unit, and one larger jump is likely. The minimum numbers of changes between karyotypes corresponded roughly one-to-one with microsatellite allele changes, which suggests very rapid chromosomal evolution. A chromosomal fission occurred in a cultured line, and a previously unknown chromosomal race was detected. All 121 diverse S. near fragariae were heterozygous but revealed only one genotype. This species too must have a low rate of sexual reproduction and few colonizations of Australia.

Distribution of the rol gene encoding the regulator of lipopolysaccharide O-chain length in Escherichia coli and its influence on the expression of group I capsular K antigens.

The rol (cld) gene encodes a protein involved in the expression of lipopolysaccharides in some members of the family Enterobacteriaceae. Rol interacts with one or more components of Rfc-dependent O-antigen biosynthetic complexes to regulate the chain length of lipopolysaccharide O antigens. The Rfc-Rol-dependent pathway for O-antigen synthesis is found in strains with heteropolysaccharide O antigens, and, consistent with this association, rol-homologous sequences were detected in chromosomal DNAs from 17 different serotypes with heteropolysaccharide O antigens. Homopolymer O antigens are synthesized by a pathway that does not involve either Rfc or Rol. It was therefore unexpected when a survey of Escherichia coli strains possessing mannose homopolymer O8 and O9 antigens showed that some strains contained rol. All 11 rol-positive strains coexpressed a group IB capsular K antigen with the O8 or O9 antigen. In contrast, 12 rol-negative strains all produced group IA K antigens in addition to the homopolymer O antigen. Previous research from this and other laboratories has shown that portions of the group I K antigens are attached to lipopolysaccharide lipid A-core, in a form that we have designated K(LPS). By constructing a hybrid strain with a deep rough rfa defect, it was shown that the K40 (group IB) K(LPS) antigen exists primarily as long chains. However, a significant amount of K40 antigen was surface expressed in a lipid A-core-independent pathway. The typical chain length distribution of the K40 antigen was altered by introduction of multicopy rol, suggesting that the K40 group IB K antigen is equivalent to a Rol-dependent O antigen. The prototype K30 (group IA) K antigen is expressed as short oligosaccharides (primarily single repeat units) in K(LPS), as well as a high-molecular-weight lipid A-core-independent form. Introduction of multicopy rol into the K30 strain generated a novel modal pattern of K(LPS) with longer polysaccharide chains. Collectively, these results suggested that group IA K(LPS) is also synthesized by a Rol-dependent pathway and that the typically short oligosaccharide K(LPS) results from the absence of Rol activity in these strains.

Structural redesign and stabilization of the overlapping tandem beta-turns of RNA polymerase II.

Peptides representing single repeat units of the carboxy-terminal domain (CTD) of RNA polymerase II (Tyr-Ser-Pro-Thr-Ser-Pro-Ser-Tyr-NH2, 1) contain overlapping Ser-Pro-Xaa-Xaa beta-turn forming sites which permit their overall structure to closely resemble members of the quinoxaline class of antitumor DNA bisintercalators. We have modified this native sequence at the i+2 positions of each beta-turn unit by substituting Gly or D-Ala in an attempt to preorganize this structure in aqueous solution. CD and NMR spectroscopic investigations confirmed the presence of type II beta-turns within each of the substituted peptides in contrast to the native sequence which contains a relatively low population of turn structure. In addition, an examination of singly substituted peptides suggests that an increase in the population of beta-turn structure within the amino-terminal Ser-Pro-Xaa-Xaa site also increased the formation of beta-turn structure in the carboxy-terminal (unmodified) Ser-Pro-Xaa-Xaa site; in comparison, substitution in the carboxy-terminal site did not influence structure in the remaining portion of the peptide. Overall, these results suggest that the structures formed could provide unique, preorganized linkers for the construction of novel DNA-interactive bisintercalators.

Functional analysis of the MAP2 repeat domain

The neuronal microtubule-associated protein MAP2 binds to microtubules via a domain near its C terminus containing a set of 3 or 4 imperfect repeats of a 31 amino acid motif. Using naturally occurring and mutated forms of the molecule containing between 1 and 4 repeats we have examined the contribution that these repeats make to MAP2 function and explored the significance of their repetition. The experiments utilised the short 3- and 4-repeat splice variants MAP2c and MAP2d that are expressed in developing neurons and in glia respectively, and mutant 1- and 2-repeat versions that were produced by using in vitro mutagenesis to remove further 31 amino acid units while leaving the rest of the molecule unaltered. The properties of these MAP2 variants were compared both with respect to their influence on microtubules in transfected non-neuronal cells and their ability to promote microtubule assembly in vitro. We found that each of the known effects of MAP2, including the bundling of microtubules and induction of process formation in living cells, are expressed by the 1-repeat form MAP2c3, which contains only the third repeat (R3). A second 1-repeat form, MAP2c4, which contains only R4, interacts more weakly with tubulin in vitro and does not bind to microtubules in transfected cells. The microtubule-related properties of MAP2 thus arise mainly from a single predominant repeat unit, R3. In vitro assembly experiments showed that the primary effect of all the repeats is to lower the critical concentration of tubulin required for microtubule assembly but that they differ greatly in potency. The results did not reveal a separate function related to the repetition of the repeat motifs, but instead suggest that its purpose is to tailor the efficiency of MAP2 to the cellular environment in which it has to function.

Expression of Shigella dysenteriae serotype 1 O-antigenic polysaccharide by Shigella flexneri aroD vaccine candidates and different S. flexneri serotypes

The potential utility of Shigella flexneri aroD vaccine candidates for the development of bi- or multivalent vaccines has been explored by the introduction of the genetic determinants rfp and rfb for heterologous O antigen polysaccharide from Shigella dysenteriae serotype 1. The serotype Y vaccine strain SFL124 expressed the heterologous antigen qualitatively and quantitatively well, qualitatively in the sense of the O antigen polysaccharide being correctly linked to the S. flexneri lipopolysaccharide R3 core oligosaccharide and quantitatively in the sense that typical yields were obtained, with ratios of homologous to heterologous O antigen being 4:1 for one construct and 1:1 for another. Moreover, both polysaccharide chains were shown to be linked to position O-4 of the subterminal D-glucose residue of the R3 core. In contrast to the hybrid serotype Y SFL124 derivatives, analogous derivatives of serotype 2a vaccine strain SFL1070 did not elaborate a complete heterologous O antigen. Such derivatives, and analogous derivatives of rough, O antigen-negative mutants of SFL1070, formed instead a hybrid lipopolysaccharide molecule consisting of the S. flexneri lipid A R3 core with a single repeat unit of the S. dysenteriae type 1 O antigen. Introduction of the determinants for the S. dysenteriae type 1 O antigen into a second serotype 2a strain and into strains representing other serotypes of S. flexneri, revealed the following for the expression of the heterologous O antigen: serotypes 1a, 1b, 2a, and 5a did not produce the heterologous O antigen, whereas serotypes 2b, 3a, 3b, 4a, 4b, 5b, and X did.

A mutation hotspot in the chloroplast genome of a conifer (Douglas-fir: Pseudotsuga) is caused by variability in the number of direct repeats derived from a partially duplicated tRNA gene.

We determined the DNA sequence of a 2.7-kb cpDNA XbaI fragment from douglas-fir [Pseudotsuga menziesii (Mirb.) Franco]. RFLPs revealed by the 2.7-kb XbaI clone were observed to vary up to 1 kb among species within the genus Pseudotsuga and up to 200 bp among trees of P. menziesii. The polymerase chain reaction (PCR) allowed the locus of polymorphism to be identified, and the variable region was then sequenced in a second Douglas-fir tree, a single tree of a related species, Japanese Douglas-fir (P. japonica), and in a species lacking a mutation hotspot in the region, Pinus radiata (Monterey pine). The locus of polymorphism is characterized by hundreds of base pairs of imperfect, tandem direct repeats flanked by a partially duplicated and an intact trn Y-GUA gene. The duplication is direct in orientation and consists of 43 bp of the 3' end of trnY and 25 bp of its 3' flanking sequence. Tandem repeats show high sequence similarity to a 27-bp region of the trnY gene that overlaps one end of the duplication. The two trees of Douglas-fir sequenced differed by a single tandem repeat unit, whereas these trees differed from the Japanese Douglas-fir sequenced by approximately 34 repeat units. Repetitive DNA in the Pseudotsuga cpDNA hotspot was most likely generated at the time of the partial trnY gene duplication and these sequences expanded by slipped-strand mispairing and unequal crossing-over.

Dielectric investigations on secondary relaxation of polyarylates: comparison of low molecular models and polymeric compounds

This paper focuses on the secondary relaxation (γ-relaxation) behaviour of a set of polyarylates and of the corresponding model systems composed of a single repeat unit and end-groups. The common feature of the compounds is that they contain short dipolar groups (O, CO, COO, SO 2) between successive phenyl units. The method of analysis was dielectric spectroscopy. The aim of the investigations was to characterize the motions connected with this relaxation and their dependence on the chemical structure. It was found that all systems display γ-relaxations which have many features in common, such as for instance the activation energy. Yet differences also exist, for instance in terms of the relaxation strength and the relaxation time distribution, which reveal the impact of the actual chemical structure on the relaxation behaviour. The general conclusion is that the γ-relaxation has to be connected both with inter- and intramolecular motions, the correlation length of which cannot be much larger than the scale set by a single repeat unit.

The cDNA sequence encoding an annexin from Medicago sativa.

Annexins are a family of at least 13 calcium-binding proteins in higher eukaryotes. They share the common features of binding phospholipids in a Ca2+-dependent manner and contain a 4or 8-fold repeated sequence of about 70 amino acid residues termed the annexin repeat. An N-terminal domain is of greater variability and may confer specific functions for each type. At least some of the annexins seem to be differentially expressed with respect to cell proliferation (Schlaepfer and Haigler, 1990). However, the biological function of the annexins is still not clearly defined. Proposed roles include exocytosis and membrane trafficking (Creutz, 1992; Gruenberg and Emans, 1993), inhibition of phospholipase Az (Haigler et al., 1987), and mitogenic signaling (Haigler et al., 1987). Until now, plant annexins have been characterized biochemically and by partial protein sequences (Boustead et al., 1989; Smallwood et al., 1990; Blackbourn et al., 1992; Andrawis et al., 1993). In cotton, an annexin-containing protein fraction was found to inhibit callose synthase in vitro (Andrawis et al., 1993). In addition, a 95-bp PCR fragrnent was isolated from tomato that was used as a probe in northern analysis to detect varying transcript levels in different plant organs (Smallwood et al., 1992). To obtain protein phosphatase genes (Pirck et al., 1993) from a Medicago sativa somatic embryo cDNA library (Hirt et al., 1991), we found that one of the cDNA inserts (AnnMs, see Table I) potentially encoded a protein with similarity to different animal annexins. A homology search at the NCBI with the BLAST net service showed 30 to 37% identity with AnnMs to various animal annexins. Considerable similarity (74%) was obtained between AnnMs and an unpublished Arabidopsis cDNA fragment potentially encoding an annexinlike protein (accession no. 218518). Comparisons with the published plant partial protein sequences revealed significant similarities between the single annexin repeat units. The tomato 28-kD fragment of p35.5 (Smallwood et al., 1990), for example, has 51% identity with repeat I1 of AnnMs. In contrast, comparison of the four AnnMs intemal repeat sequences showed only approximately 20% identity. Similar results were obtained when various animal annexins were analyzed.

Defective Marek's Disease Virus DNA Contains a Gene Encoding a Potential Nuclear DNA Binding Protein and a HSV a-like Sequence

Four RNA transcripts from chicken embryo fibroblast cells infected with Marek's disease virus (MDV) strain 281MI/1 hybridized to the 4-kbp MDV replicon DNA. In an attempt to identify open reading frames coding for the four transcripts, we determined the nucleotide sequences of 4-kbp replicon DNA (represents a single monometic repeat unit of defective MDV genome). Computer analysis indicates that the 4-kbp MDV replicon DNA contains two intact open reading frames (ORFs) with common promoter regulatory elements. ORF-A codes for a putative 204 amino acid protein that shares 21 and 36% amino acid sequence identity to nuclear DNA binding proteins such as the EBNA-1 of Epstein-Barr virus and galline, a chicken sperm histone protein, respectively. ORF-B encodes for a potential 350 amino acid protein, which did not show any significant amino acid sequence identity to known protein sequences within Swiss-Protein data base. ORF-B may, therefore, encode a MDV specific protein. The 5′-region of MDV replicon DNA revealed seven reiterated copies of an 11-bp motif sharing 8 out of 11 nucleotide sequence identity to DR2 elements of the herpes simplex virus strain USA-8 a sequence.

The rRNA-encoding DNA array has an altered structure in topoisomerase I mutants of Saccharomyces cerevisiae.

All the chromosomes from isogenic TOP1 and top1 strains have similar mobility on pulsed-field gels except for chromosome XII, which fails to migrate into the gels in top1 mutants. Chromosome XII contains the tandem repeats of rRNA-encoding DNA (rDNA). When a segment of chromosome XII containing only rDNA is transferred to chromosome III by a recombination event, chromosome III fails to enter a pulsed-field gel in extracts from top1 strains, indicating that the aberrant migration of chromosome XII in top1 mutants is caused by the presence of rDNA. Failure of chromosome XII to migrate into a pulsed-field gel occurs only in preparations from exponentially growing top1 cultures and not in preparations from stationary-phase top1 cultures. rDNA from a top1 strain does enter the gel if it is cut with an enzyme (Pst I) that cuts the tandem rDNA array into single 9-kb repeat units, indicating that more than a single repeat unit is required to maintain the aberrant structure.

Acyclic diene metathesis (ADMET) copolymerization. Controlled diene insertion in poly[(hexamethyltrisiloxanediyl)butenylene

The single repeat unit insertion of 4,4'-di-trans-l-propenylbiphenyl(4) into poly( (1,1,3,3,5,5- hexamethyl-l,3,5-trisiloxane-l,5-diyl) butenylene) (3) via acyclic diene metathesis (ADMET) copolymerization catalyzed by ((CF~)~CH~COIO(N-~,~-C~H~-~-P~~)W==CHC(CH~)~P~ (1) is presented. The cross metathesis condensation of an unsaturated polymer with an aromatic diene monomer allowed for random and isolated insertion of the biphenylene linkages throughout the polycarbosiloxane. In addition to cometathesis reactions, the copolymer 5 also continued to condense ethylene and grow in molecular weight. The copolymer was characterized by lH NMR and quantitative 13C NMR spectroscopy and UV spectrophotometry. Molecular weights were determined by quantitative 13C NMR and GPC. The copolymer 5 underwent uninitiated cross- linking under an inert atmosphere and degraded after prolonged exposure to air under ambient conditions. Thermal stabilities of the polymer starting material, copolymer, and degraded products are compared.

Mutation of human short tandem repeats.

A total of 20,000 parent-offspring transfers of alleles were examined through the genotyping within 40 CEPH reference families of 28 short tandem repeat polymorphisms (STRPs) located on chromosome 19. Forty-seven initial mutation events were detected in the STRPs using DNA from transformed lymphoblastoid cell lines, but less than half (39%) could be verified using DNA from untransformed cells. None of the cases where three alleles were observed in a single individual could be verified using DNA from untransformed cells. The average mutation rate for the chromosome 19 STRPs after correction for events which would not be detectable as Mendelian errors was 1.2 x 10(-3) per locus per gamete per generation. This rate may have been inflated by somatic as opposed to germline events. Observed mutation rates for individual STRPs ranged from 0 to 8 x 10(-3). The average mutation rate for tetranucleotide STRPs was nearly four times higher than the average rate for dinucleotide STRPs. For determination of the mode of mutation, events involving STRPs on other chromosomes were also examined. Of the events which were verified using DNA from untransformed lymphocytes or which were likely among those for which DNA from untransformed cells was not available: none were located at the sites of meiotic recombination, 91% involved the gain or loss of a single repeat unit, and 15 occurred in the male germline compared to 4 in the female germline (p = 0.01).

The flax ribosomal RNA-encoding genes are arranged in tandem at a single locus interspersed by ‘non-rDNA’ sequences

The ribosomal RNA (rRNA)-encoding genes (rDNA) in flax, estimated to be present in about 2400 copies per diploid nucleus, have been reported as a single homogeneous repeat unit of 8.6 kb. In situ hybridization analysis indicated that these genes were located at a single site on one pair of chromosomes. However, an analysis of a flax variety, CI 1303, has revealed heterogeneity in the intergenic spacer of the rDNA repeat unit. A genetic analysis of rDNA inheritance in two flax lines, Stormont Cirrus and CI 1303, has again supported the observation that there is a single rDNA locus in this plant species. Screening of four different genomic libraries made in methylation-sensitive and -insensitive systems, and the analysis of 40 phage clones, demonstrate a much higher number than that expected of junctions between rDNA and non-rDNA. Direct evidence of rRNA-encoding genes being present in tandem comes from a few phage clones that contain more than two rDNA repeats. The evidence presented here indicates that rDNA, although present at a single locus in tandem arrays, may be interrupted frequently by other non-rDNA sequences, thus giving rise to questions about their organization into long tandem arrays.

X-ray powder diffraction on oligomeric and polymeric permethylpolyazine

X-ray powder patterns on oligomeric and polymeric permethylpolyazines are reported. These materials are largely amorphous but diffraction spots are observed which can be interpreted in an orthorhombic unit cell. The cell parameters for the polymer describe the volume of a single repeat unit in the all trans conformation, as previously implied from IR and NMR data. In contrast, a packing analysis of cis conformations leads to unit cells larger than experimentally observed.

Complete human rDNA repeat units isolated in yeast artificial chromosomes

Human ribosomal DNA has been inferred to be organized in tandem repeat units of 44 kb, of which only 13 kb is transcribed into preribosomal RNA. Unfortunately, it has remained difficult to examine the intact repeat structure directly, because even a single repeat unit is too large to be accommodated in conventional cloning systems. Here we report the isolation of intact repeat units using yeast artificial chromosomes as a cloning tool. With a spacer sequence specific to human ribosomal DNA used as a probe, 27 clones were identified among 17,000 YACs (about 0.7 genomic equivalent of total human DNA). Fourteen clones contained only a small portion of rDNA; the other 13 contained most or all of the rDNA repeat unit, and 8 of those were studied in further detail. They contained 1 to 1.5 repeat units of rDNA with all of the expected EcoRI and HindIII fragments. These clones provide possible starting material for the analysis of expression of a single unit of rDNA. Unexpectedly, however, only the four smaller clones (70 to 90 kb) were completely composed of standard rDNA sequences; four larger clones (up to 950 kb in length) contained additional “non-rDNA” sequences, at either one or both ends of the repeat unit. Analysis of these atypical rDNA clones suggests that their inserts either are scattered in the genome or are localized in a nucleolar organizer region that is more complex than previously recognized.

Enzymatic synthesis of DNA probes complementary to a human variable number tandem repeat locus

Both cloned and synthetic DNA probes complementary to human variable number tandem repeat (VNTR) loci have been used to detect restriction fragment length polymorphism. In this report, we describe an approach for the enzymatic synthesis of a DNA probe complementary to one VNTR locus. The probe is produced by annealing short synthetic oligonucleotides comprising single repeat units and enzymatically ligating them into a polymeric DNA probe. In HinfI digests of human genomic DNA separated by agarose gel electrophoresis, this ligated oligonucleotide probe (LOP) detects multiple polymorphic loci in the range of 3–23 kb producing highly informative DNA fingerprint patterns when different individuals are compared. The hybridization pattern is very stable even under high-stringency wash conditions. The LOP is more easily generated than cloned VNTR probes and is totally synthetic, avoiding problems associated with cloned probes including bacterial growth and maintenance as well as in vitro labeling.

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 BR1gamma 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 BR1gamma 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 ribosomal DNA transcription unit of the house cricket, Acheta domesticus.

A composite map representing a single ribosomal DNA repeat unit of the house cricket, Acheta domesticus, was constructed from overlapping cloned fragments. Sites in the repeat unit for nine restriction enzymes were mapped. R-loop mapping of sequences coding for 18 S and 28 S RNA demonstrates that the 58-kb ribosomal DNA repeat unit contains a novel-sized internal transcribed spacer of 8.4 kb. The existence of this large spacer was confirmed in genomic DNA, most if not all of the genomic repeat units containing such a spacer. A 15- to 17-kb ribosomal RNA precursor transcript is synthesized as predicted on the basis of the size of the internal transcribed spacer. The 5.8 S RNA gene is localized to a 1-kb sequence immediately 5' to the 28 S gene. The coding regions examined contain no intervening sequences analogous to those described within ribosomal DNA of other eukaryotes. Only 11% of the repeat unit codes for mature ribosomal RNA, while the remainder is nontranscribed (71%) and transcribed (18%) spacer DNA.

Herpes simplex virus infection generates large tandemly reiterated simian virus 40 DNA molecules in a transformed hamster cell line.

When the simian virus 40 (SV40)-transformed Syrian hamster cell line Elona is infected with herpes simplex virus type 1, an excessive amplification of SV40-specific DNA sequences occurs. Analysis of total DNA from herpes simplex virus-infected cells revealed that amplified DNA sequences were present predominantly in a high-molecular-weight form, consisting of a tandem array of many unit-length SV40 DNA molecules. Repeat units of amplified DNA were found to be very similar to standard SV40 DNA as was shown by restriction analyses, except for a small deletion close to the origin of replication, which could also be detected in the chromosomal DNA of uninfected cells. A procedure, devised for selective enrichment of amplified SV40 DNA molecules from the bulk of cellular and herpesviral DNA, allowed molecular cloning of single repeat units and nucleotide sequence analysis of the relative genomic region.