Kbp In Length Research Articles

Purification of Cowdria ruminantium organisms for use in genome analysis by pulsed-field gel electrophoresis.

Cowdria ruminantium is an obligate intracellular rickettsial pathogen which is responsible for a tick-borne disease of domestic and wild ruminants called heartwater or cowdriosis. Although several genes have been cloned and partially sequenced, the genome size, gross structure, and organization of the C. ruminantium genome is unknown. Genome analysis of the organism has been hindered because it is difficult to obtain C. ruminantium DNA free from contaminating host cell DNA, and this probably accounts for the lack of genome size data for this organism. In this study we investigated several methods for purifying C. ruminantium from bovine cellular contaminants and organisms of a relatively high purity were obtained. These were used to prepare Cowdria DNA which was analyzed by pulsed-field gel electrophoresis (PFGE) and which revealed a genome approximately 1900 kbp in length plus an additional extra-chromosomal fragment migrating with an apparent size of 815 kbp. This is the first time that the genome size of C. ruminantium has been determined and the first demonstration of an extrachromosomal element.

Expression cloning and characterization of a renal organic anion transporter from winter flounder

The cDNA coding for a renal p-aminohippurate (PAH) transporter from winter flounder ( Pseudopleuronectes americanus), designated fROAT, was cloned by functional expression in Xenopus laevis oocytes. fROAT is approximately 2.8 kbp in length and encodes a protein of 562 amino acids, related to the rat renal organic anion transporter ROAT1/OAT1 and the organic cation transporters OCT1 and OCT2. In oocytes, fROAT mediated probenecid-sensitive PAH uptake, with a K m for PAH of about 20 μM, and inhibited by external glutarate (GA) (1 mM). The functional characteristics suggest that fROAT is the basolateral PAH/dicarboxylate exchanger of the flounder kidney.

A slot-blot hybridisation method for screening somatic hybrids

A slot-blot method is described that provides rapid and reliable identification of somatic hybrids. Biotin-labelled probes of defined length were prepared from genomic DNA, and calibrated in conjunction with target DNA to allow for differing amounts of biotin incorporation and hybridisation efficiency. The presence of probe DNA was visualised by chemiluminescence detection. Probes of 1 to 5 kbp in length gave optimal discrimination among target DNAs from different potato species. The advantages of this method over existing ones are described.

A Comparison of Electrophoretic Resolution for Snapshot and Finish-Line Imaging

Finish-line imaging, in which DNA or other macromolecules are detected after electrophoresis for a constant distance, usually improves resolution compared to snapshot imaging, in which molecules are electrophoresed for a constant time in an apparatus of comparable dimensions. Resolving power, which is an objective measure of the ability of different separatory methods to detect closely spaced molecular species, can be used to compare directly the performance of systems employing both snapshot and finish-line imaging [E. A. Ribeiro and J. C. Sutherland,Anal. Biochem.210, 378–388 (1993)]. Experimentally determined values of resolving power are influenced both by the method of imaging (snapshot vs finish-line) and by instrument-specific factors that affect resolution. Previous comparisons of the resolving power obtained with finish-line and snapshot imaging involved data sets acquired by different instruments with different instrumental resolutions. To reduce the influence of instrumental effects, we constructed a scanning laser fluorometer that can measure both snapshot and finish-line images of fluorochrome-labeled DNA. Snapshot and finish-line images of a DNA sample containingHaeII restriction fragments of the DNA from bacteriophage T7, which range in length from 474 to 6514 base pairs, were obtained under otherwise identical electrophoretic conditions. Snapshot and finish-line imaging give similar resolving powers for DNA molecules up to about 1.5 kbp long. For both imaging modes, maximum resolving power was achieved for DNA molecules between 2 and 3 kbp in length. For larger DNA molecules, finish-line imaging provided higher resolving power. The ratio of the resolving power of finish-line images to that of snapshot images increased monotonically as a function of DNA length. For the longest restriction fragments studied (6514 bp), the resolving power for finish-line images exceeded that of snapshot images by about 50%.

Gene targeting of a plasmid-borne sequence to a double-strand DNA break in Drosophila melanogaster.

We report an efficient and specific gene targeting method for transforming the germ line of Drosophila melanogaster. The targeting occurs during the repair of a double-strand DNA break that is induced at the white locus by the excision of a P transposable element. The break is repaired when homologous sequence is copied from a plasmid injected into the Drosophila embryo. The procedure efficiently integrates DNA into the targeted locus of the Drosophila genome. Heterologous sequence of up to 13 kbp in length can be inserted, permitting the intergration of entire genes into a common genomic site for further study.

Genomic organization of ribosomal RNA genes inBromus(Poaceae)

Restriction site maps of the rDNA genes of nine Bromus species are described. The rDNA repeat units ranged from 8.2 to 11.1 kbp in length. Intraspecific length variation was observed in the BamHI digestions in three of the nine species. Restriction site variation was observed mainly in the intergenic spacer (IGS) but was also detected in the coding region. A unique KpnI site was present in the IGS of Bromus tectorum and Bromus sericeus (subgenus Stenobromus); in addition, B. sericeus contained an extra EcoRI site. An additional DraI site was observed in the IGS of Bromus trinii (subgenus Neobromus). A BstEII site in the IGS, common to seven of the species, was absent in B. tectorum and B. sericeus. In the coding region, a 2.1-kbp BstEII fragment was present in four subgenera represented by Bromus inermis and Bromus erectus (subgenus Festucaria), Bromus marginatus and Bromus carinatus (subgenus Ceratochloa), B. tectorum and B. sericeus (subgenus Stenobromus), and B. trinii (subgenus Neobromus); a similar fragment of only 1.1 kbp was present in Bromus mollis and Bromus arvensis (subgenus Bromus). An additional BamHI site was present in the coding region of B. erectus. Ribosomal DNA data suggested that B. mollis and B. arvensis (subgenus Bromus) are genetically isolated from the other subgenera, which showed a derived relationship. Restriction site mapping of the rDNA genes could provide useful molecular data for species identification and population and evolutionary studies in Bromus. Key words : Bromus, ribosomal DNA, restriction maps, evolutionary relationships.

The relative contributions of dispersion and diffusion to band spreading (resolution) in gel electrophoresis.

DNA of approximately 2 kbp in length was previously found not to diffuse significantly in 1-1.5% agarose gels in the absence of an electric field, but to disperse during electrophoresis (Yarmola, E., Chrambach, A., Electrophoresis 1995, 16, 345-349). Accordingly, a process distinct from diffusion, and responsible for band spreading with migration time in gel electrophoresis, was defined as dispersion. Correspondingly, the diffusion coefficient, D(diff), was distinguished from a dispersion coefficient, D(disp). For DNA of approximately 1, 2 and 3 kbp, D(diff) and D(disp) were measured in agarose gel electrophoresis (1.0% SeaKem GTG). In that order of DNA length, D(disp)/D(diff) was found to increase from 5 to 15 to 45, showing that with increasing DNA length, time-dependent band spreading, and thus resolution in gel electrophoresis, is governed predominantly by dispersion, not diffusion. It is assumed that the essential part of electrophoretic dispersion is due to entanglement of the DNA molecule in the gel. Indirect evidence for such an entanglement derives from the observation of peak asymmetry and its interpretation by the Giddings-Weiss model.

Comamonas acidovorans UCC61 catabolizes o-phthalate via a 4,5-oxygenation pathway that is encoded on a 70 kbp section of plasmid pOPH1 bounded by directly repeated sequences

Summary: Twenty-three bacteria capable of growing with o-phthalate as sole carbon source were isolated from sewage sludge. One of these, named UCC61, was identified as a strain of Comamonas acidovorans and was selected for further study. UCC61 was found to carry a single plasmid (named pOPH1) of about 270 kbp in size. The phthalate-utilizing phenotype of UCC61 was unstable and could be lost either by complete curing of pOPH1, or by the deletion of a specific 70 kbp segment from pOPH1. This segment, termed the Pht element, was extensively restriction mapped (using four restriction endonucleases), and was found to be flanked by directly repeated sequences greater than 1-9 kbp in length. Data from comparative restriction analysis of pOPH1 and its deleted derivative were consistent with a deletion mechanism that involves homologous recombination between the direct repeats. UCC61 was found to catabolize o-phthalate via 4,5-oxygenation, dehydrogenation and decarboxylation to protocatechuate. These three activities were encoded within the Pht element, with the genes clustered near the right-hand terminus. Protocatechuate metabolism was chromosomally encoded, as also was the ability to catabolize the m- and p-isomers of phthalate.

Presence of a Chloroplast DNA Sequence in an Autonomous Circular DNA Molecule in Cultured Rice Cells (<italic>Oryza sativa</italic> L.)

Sequence analysis of twelve DNA fragments, which had previously been found to be extensively amplified in suspension-cultured rice cells, revealed that two of them, isolated on plasmids designated pE10 and pE11, have sequences identical to distinct regions of chloroplast DNA (ct-DNA). Both sequences are part of an extrachromosomal circular DNA molecule (ECD). The molecular structure of the ECD was investigated by a combination of restriction analysis, standard and pulsed-field gel electrophoresis, hybridization with ct-DNA probes and amplification by the polymerase chain reaction in the presence of oligonucleotide primers homologous to selected regions of rice ct-DNA. The results showed that a continuous and unrearranged stretch of ct-DNA from the long single-copy region, of at least 28 kbp in length, is present in the ECD. It was estimated that the number of copies of the ECD in cultured cells was almost equivalent to that of ct-DNA molecules in rice leaves, while the ratio of ECD to ct-DNA molecules in the cultured cells was approximately 200:1.

DNA probe specific to Rhizoctonia solani anastomosis group 8

A DNA probe was developed which hybridizes strongly with DNAs of all isolates of Rhizoctonia solani anastomosis group 8 (AG-8) tested. The probe hybridized only weakly or not at all with DNA of isolates from other anastomosis groups. The results presented here indicate that the probe recognizes a dispersed repeated DNA element in AG-8 isolates and that genetic variation occurs within AG-8. Preliminary experiments show that the repeated unit is at least 1·8 kbp in length and that sequence variation occurs between different repeat units. The probe has the potential to further our understanding of the population dynamics of bare patch disease in cereal crops.

Unclassified Poxvirus: Characterization and Physical Mapping of Cotia Virus DNA and Location of a Sequence Capable of Encoding a Thymidine Kinase

DNA from Cotia virus, an unclassified poxvirus, was mapped by overlapping fragment analysis using the restriction endonucleases HindIII, Pst I, BamHI, XhoI, SalI, and SmaI. The linear genome was 177 kbp in length and possessed inverted terminal repeats and cross-links. A Cotia virus thymidine kinase (TK) gene was detected and mapped to about 74 kbp from the left end of the genome using degenerate oligonucleotide probes Nucleotide sequencing of the TK gene revealed an open reading frame (ORF) that encoded a peptide of 178 amino acids, An A/T-rich sequence, TAA AAA TGA ATA AATA, and a transcription termination signal, TTTTTGT, were revealed upstream and just downstream of the ORF, respectively, consistent with the characteristic features of an early poxvirus gene. Cotia virus resembles swinepox virus based on the restriction profiles generated by endonucleases and shares amino acid sequence similarity with orthopox, leporipox, Yaba, and fowlpox viruses.

Structure of the human C7 gene and comparison with the C6, C8A, C8B, and C9 genes.

The seventh component of complement is a single chain plasma glycoprotein that is involved in the cytolytic phase of complement activation. We have determined the structure of the C7 gene, which is encoded by 18 exons whose sizes vary from 56 to 244 bp. For the most part, the exons do not correspond to the protein homology units. However, two intron/exon boundaries occur at junctions between different functional parts of the protein. The first is at a site between the end of the C9 homology unit and the carboxyl-terminal extension which is also a feature of C6. The second of these boundaries occurs between the regions encoding two pairs of cysteine-rich modules (the short consensus repeats and the factor I modules) located in the carboxyl-terminal part of C7. In contrast to the exons, the introns range considerably in size from 0.5 to 8.5 kbp. The complete analysis indicates that the gene encoding C7 is approximately 80 kbp in length. We show here that the C7 gene is highly homologous to that for C6, and also to C8A, C8B, and C9, confirming and extending the published data. With the exception of exon 1, all intron/exon boundaries are preserved with respect to phase when compared with C6.

Evolutionary Biology of Trillium and Related Genera (Trilliaceae) I. Restriction Site Mapping and Variation of Chloroplast DNA and its Systematic Implications

Abstract A physical map of chloroplast DNA (cpDNA) was constructed for Trillium kamtschaticum using three restriction enzymes, Apal, Pstl and Xhol. The chloroplast genome is 154.5 kbp in size with each portion of the inverted repeat 26.5 kbp in length, separated by large (82.7 kbp) and small (18.8 kbp) single copy regions. The cpDNA of T. kamtschaticum is similar in structure to the cpDNAs of most angiosperms thus far studied. Chloroplast DNA variation in 25 taxa of Trillium was investigated using 14 restriction enzymes, with Paris tetraphylla as an outgroup. Phylogenetic analysis showed that sessile‐flowered species (subgenus Phyllantherum) form a monophyletic group. Pedicellate‐flowered species do not form a monophyletic group, and belong to at least three different groups. T. kamtschaticum, an East Asian diploid species, was found to be most closely related to a group of species from eastern North America.

Gene structure and cell type-specific expression of the human ATP synthase α subunit

The gene structure of the human ATP synthase α subunit (hATP1) was determined by cloning and sequencing. This gene is approximately 14 kbp in length and contains 12 exons interrupted by 11 introns. Mapping of the clones of hATP1 and Southern blot analysis of the genomic gene showed that there were a single copy of bona fide hATP1 gene and two pseudogenes. Primer extension and S1 mapping analysis showed the presence of multiple transcription initiation sites of the hATP1 gene. No TATA box or CAAT box was found near the transcription initiation sites. Comparison with the bovine gene showed that the 5′-flanking region of the hATP1 gene has an unconserved guanine-cytosine (GC) rich region, including several binding motifs of transcriptional factors, such as Sp1, AP-2, and GCF. By functional assay of gene expression, the basal promoter activity was located near the GC rich region. Comparison of the 5′-upstream region of the hATP1 gene with those of the genes for bovine ATP synthase α, human β, and human γ subunits indicated three common sequences, suggesting that putative cis-elements coordinate the expressions of the three subunit genes for the ATP synthase. The enhancer activities derived from the 5′-deletion mutants of a hATP1-CAT chimeric gene were different in cell lines from four different human tissues, suggesting the existence of cell type-specific gene regulation.

Diversity of tandemly repetitive sequences due to short periodic repetitions in the chromosomes of Candida albicans

In a previous study, a repeated sequence, RPS1, was cloned from the genomic DNA of Candida albicans. It was 2.1 kb in length and was tandemly repeated in a limited region of almost all of the chromosomes. In this study, we examined and characterized the diversity of the repeating structure of the RPS units were of 2.1, 2.3, 2.5, and 2.9 kbp in length after digestion of the genomic DNA with SmaI and 2.1 and 2.3 kbp after digestion with PstI, with the differences being multiples of approximately 0.2 kbp. Moreover, one or two types of RPS unit were present specifically on each chromosome. We cloned 14 RPS units from the mixed DNA of chromosomes 1 and 2 and 59 RPS units from chromosome 6. These RPS units were classified into four types by their SfiI digestion profiles and chromosomal origins. Sequence comparisons revealed a tandem arrangement of internal, small repeating units of 172 bp. This unit of repetition was designated alt (C. albicans tandem repeating unit). The size of RPS units was variable, with sizes representing a series of increments of approximately 0.2 kbp that corresponded to the alt sequence. By contrast, the sequences other than the tandem repeats of alts were highly conserved, with homology of more than 98% among all cloned RPS units. These results suggested that RPS plays an important role in the organization and function of the chromosomes of C. albicans even though the actual function of RPS has not yet been clarified. Structural features of RPS that contains the repeated alt sequence are discussed in relation to human alpha-satellite DNA with its tandem repeats of about 170 bp that are similar in size to alt, the repetition of which is responsible for the variations in the size of the higher-order repeats.

Alternate Polyadenylation in Rodent Cells Results in Two Differentially Expressed Cyclin B1 mRNAs

Cyclin B1 is required for the G2/M transition. In HeLa cells, the level of both its protein and mRNA varies dramatically through the cell cycle. In this study we examine the expression of cyclin B1 mRNA in Chinese hamster ovary cells and 3.7, a transformed rat embryo fibroblast line. In contrast to HeLa cells in which a single transcript is seen, both of these lines express two distinct transcripts of approximately 1.4 and 2.7 kbp in length which appear to be made using different polyadenylation sites. In CHO cells, the level of the upper band varies modestly through the cell cycle while the lower band remains almost constant. In contrast to HeLa cells, CHO cells in G1 express a significant amount of cyclin B1 mRNA. In synchronized 3.7 cells, the level of both transcripts is very low in G1 and rises in parallel as cells progress through S and G2, but the longer transcript disappears faster as cells reenter G1.

Identification of a Novel Drosophila Gene Encoding a Cdc2-Related Protein Kinase

We have identified a novel gene encoding a putative protein kinase from a Drosophila genomic library. The gene, about 2 kbp in length, consists of four exons and codes for a protein of 349 amino acid residues. The deduced sequence shows significant similarity to various kinases, especially to a subgroup of Ser/Thr kinases related to Cdc2 kinase; thus, the gene was termed Dcdrk (Drosophila cdc2-related kinase gene). Among the kinases examined, mammalian galactosyltransferase-associated 58 kDa protein kinase showed the highest homology (about 50% identity in the kinase domain) to Dcdrk kinase. Northern blot analysis revealed that the Dcdrk mRNA is expressed throughout development in nearly constant amounts. Moreover, a whole mount in situ hybridization experiment showed that the Dcdrk mRNA is ubiquitously distributed in almost all embryonic cells and tissues, suggesting a universal function of Dcdrk, possibly in cell cycle regulation.

Chromosomal replication initiates and terminates at random sequences but at regular intervals in the ribosomal DNA of Xenopus early embryos.

We have analysed the replication of the chromosomal ribosomal DNA (rDNA) cluster in Xenopus embryos before the midblastula transition. Two-dimensional gel analysis showed that replication forks are associated with the nuclear matrix, as in differentiated cells, and gave no evidence for single-stranded replication intermediates (RIs). Bubbles, simple forks and double Ys were found in each restriction fragment analysed, showing that replication initiates and terminates without detectable sequence specificity. Quantification of the results and mathematical analysis showed that the average rDNA replicon replicates in 7.5 min and is 9-12 kbp in length. This time is close to the total S phase duration, and this replicon size is close to the maximum length of DNA which can be replicated from a single origin within this short S phase. We therefore infer that (i) most rDNA origins must be synchronously activated soon in S phase and (ii) origins must be evenly spaced, in order that no stretch of chromosomal DNA is left unreplicated at the end of S phase. Since origins are not specific sequences, it is suggested that this spatially and temporally concerted pattern of initiation matches some periodic chromatin folding, which itself need not rely on DNA sequence.

Characterization of a virulent Lactobacillus sake phage PWH2

A virulent bacteriophage PWH2 was isolated from fermented sausage. Out of 14 strains of Lactobacillus sake and 5 strains of L. curvatus tested as potential hosts, only L. sake Ls2 was sensitive. The plaques had a diameter of 0.5-1 mm. One-step growth kinetics of bacteriophage PWH2 revealed the following characteristics: a latent period of 1.5 h, a rise period of 1 h and a burst size of 110 (+− 10) phages per cell. From electron micrographs it was deduced that bacteriophage PWH2 has an icosahedral head, a long non-contractile tail and a complex base plate. The genome is linear and 35 kbp in length. The structural proteins consist of three major and two minor proteins. After an infection of L. sake Ls2, isolates were obtained that were resistant to PWH2. By treatment with mitomycin C, isolate R4a was found to be lysogenic. DNA/DNA hybridisation proved homology of phage PWH2 with the chromosomal DNA of strain R4a.

Knotting of a DNA chain during ring closure.

The formation of knotted species on random ring closure of two DNAs that are 5.6 kilobase pairs (kbp) and 8.6 kbp in length was measured, and these data were used to calculate the effective DNA helix diameter as a function of sodium ion and magnesium ion concentration. In the presence of more than 50 mM magnesium ion, interactions between DNA segments appear to be attractive rather than repulsive. The free energy of formation of relaxed trefoil and figure-eight DNA knots and of supercoiled trefoil DNA knots was also evaluated.