Distribution and evolution of two satellite DNAs in the genus Beta

Summary EcoRI monomers of a highly repetitive DNA family of Beta vulgaris have been cloned. Sequence analysis revealed that the repeat length varies between 157–160 bp. The percentage of AT-residues is 62% on average. The basic repeat does not show significant homology to the BamHI sequence family of B. vulgaris that was analyzed by us earlier. Both the EcoRI and BamHI sequences are investigated and compared to each other with respect to their genomic organization in the genus Beta. Both repeats were found to be tandemly arranged in the genome of B. vulgaris in a satellite-like manner. The EcoRI satellite DNA is present in three sections (Beta, Corollinae and Nanae) of the genus, whereas the BamHI satellite DNA exists only in the section Beta. The distribution of the EcoRI and BamHI satellite families in the genus is discussed with respect to their evolution.     

Comparative restriction endonuclease analysis and molecular cloning of plastid DNAs from wild species and cultivated varieties of the genus Beta (L.)

Summary A phyletic tree of the genus Beta has been constructed based on EcoRI and PstI plastid DNA restriction patterns of eight species from three sections of the genus. In contrast to the remarkable morphological variability of the varieties of B. vulgaris the restriction patterns of the plastid DNA of this species were found to be almost identical. The comparison of plastic DNAs of B. vulgaris crassa fertile and sterile lines with 13 different restriction enzymes revealed only a single fragment polymorphism in the HindIII patterns. Hybridization analyses in the plastidal rDNA region revealed an interesting loss of an EcoRI restriction site in all cultivated B. vulgaris varieties in contrast to wild species. The results of the construction of clone banks for SalI and BamHI fragments of plastid DNA from fertile B. vulgaris crassa are reported and difficulties in the cloning of specific fragments are discussed.     

Molecular cloning of tobacco chloroplast ribosomal RNA genes

Summary Tobacco chloroplast ribosomal RNAs were shown to be hybridized with two EcoRI fragments of tobacco chloroplast DNA. These DNA fragments having molecular weights of 1.9x10⁶ and 2.8x10⁶ daltons were cloned using the bacterial plasmid pMB9 as a vector and E. coli HB101 as host bacteria. The recombinant plasmids containing either or both of these fragments were constructed and characterized.Abbreviations rRNA ribosomal RNA - EDTA ethylenediamine tetraacetic acid - SSC 0.15 M NaCl-0.015 M sodium citrate - EcoRI and HindIII restriction endonucleases isolated from E. coli RY13 and Haemophilus influenzae Rd, respectively     

DNA of Epstein-Barr virus. IV. Linkage map of restriction enzyme fragments of the B95-8 and W91 strains of Epstein-Barr Virus.

The arrangement of EcoRI, Hsu I, and Sal I restriction enzyme sites in the DNA of the B95-8 and W91 isolates of Epstein-Barr virus (EBV) has been determined from the size of the single-enzyme-cleaved fragments and from blot hybridizations that identify which fragments cut from the DNA with one enzyme contain nucleotide sequences in common with fragments cut from the DNA with a second enzyme. The DNA of the B95-8 isolate was the prototype for this study. The data indicate that (i) approximately 95 X 10(6) to 100 X 10(6) daltons of EBV (B95-8) DNA is in a consistent and unique sequence arrangement. (ii) Both termini are variable in length. One end of the molecule after Hsu I endonuclease cleavage consists of approximately 3,000 base pairs, with as many as 10 additional 500-base pair segments. The opposite end of the molecule after Sal I endonuclease cleavage consists of approximately 1,500 base pairs, with as many as 10 additional 500-base pair segments. (iii) The opposite ends of the molecule contain homologous sequences. The high degree of homology between the opposite ends of the molecule and the similarity in size of the "additional" 500-base pair segments suggests that there are identical repeating units at both ends of the DNA. The arrangement of restriction endonuclease fragments of the DNA of the W91 isolate of EBV is similar to that of the B95-8 isolate and differs from the latter in the presence of approximately 7 X 10(6) daltons of "extra" DNA at a single site. Thus, the size of almost all EcoRI, Hsu I, and Sal I fragments of EBV (W91) DNA is identical to that of fragments of EBV (B95-8) DNA. A single EcoRI fragment, C, of EBV (W91) DNA is approximately 7 X 10(6) daltons larger than the corresponding EcoRI fragment of EBV (B95-8) DNA. Digestion of EBV (W91) DNA with Hsu I or Sal I restriction endonucleases produces two fragments (Hsu I D1 and D2 or Sal I G2 and G3) which differ in total size by approximately 7 X 10(6) daltons from the fragments of EBV (B95-8) DNA. Furthermore, the EcoRI, Hsu I, and Sal I fragments of EBV (W91) and (B95-8) DNAs, which are of similar molecular weight, have homologous nucleotide sequences. Moreover, the W91 fragments contain only sequences from a single region of the B95-8 genome. Two lines of evidence indicate that the "extra" sequences present in W91 EcoRI fragment C are viral DNA and not cellular. (i) The molecular weight of the "enlarged" EcoRI C fragment of EBV (W91) DNA is identical to that of the EcoRI C fragment of another isolate of EBV (Jijoye), (ii) The HR-1 clone of Jijoye has previously been shown to contain DNA which is not present in the B95-8 strain but is present in the EcoRI C and Hsu I D2 and D1 fragments of EBV (W91) DNA (N. Raab-Traub, R. Pritchett, and E. Kieff, J. Virol. 27:388-398, 1978).     

Partial structure of the human H-protein gene

In a span of approximately 13 kb, two genomic fragments showing an obvious overlap encoded the 1,192 base pair (bp) cDNA sequence for human H-protein. Because of the close similarity in the genomic organization comprised of five exons to that for the chicken H-protein gene, this region was assigned to the true H-protein gene in human. Primer extension analysis suggested and S1 protection analysis confirmed that at least one additional exon for the 74 bases-long 5' untranslated region in H-protein mRNA exists as the presumable first exon. None of the 5.0- and 5.5-kb SacI and the 5.2-kb EcoRI fragments which were undetectable in the genomes of patients with nonketotic hyperglycinemia was included in the true H-protein gene.     

Catabolic instability,plasmid gene deletion and recombination in Alcaligenes sp. BR60

An Alcaligenes sp. BR60, isolated from surface runoff waters of the Hyde Park industrial landfill, contained a novel 85 kb catabolic plasmid (pBR60) functional in 3-chlorobenzoate (3Cba) degradation. The plasmid exhibited a spontaneous 3.2% frequency of deletion of a 14 kb fragment specifying 3Cba degradation. The deletion mutant BR40 and mitomycin C cured strains were not able to grow on 3Cba and had reversion frequencies of less than 10^-10 cell^-1 generation^-1. Transformation or conjugation of pBR60 into cured strains restored catabolic activity. An EcoRI, BgIII, HindIII and SaII restriction map of the deletion region was constructed, and EcoRI and HindIII fragments spanning the deletion region of the plasmid were cloned in pUC18. Conjugation of resistance plasmid R 68.45 into Alcaligenes sp. BR60, with selection on antibiotics, resulted in the elimination of pBR60 and maintenance of unaltered R68.45. In 30% of the exconjugants, 3Cba degradative capacity was retained, although variation in the regulation of 3Cba degradation was observed in these strains. Hybridization of deletion region fragments to BgIII digested total DNA of BR60 and the R68.45 cured exconjugants revealed the presence of pBR60 deletion region sequences in the chromosome of exconjugants. Hybridization also revealed a repeated sequence flanking the deletion region of pBR60. Selection on 4-chlorobenzoate as a sole source of carbon and energy resulted in the isolation of 4Cba⁺ mutants of Alcaligenes sp. BR60.Abbreviations 3 and 4 Cba chlorobenzoic acid isomers and growth phenotypes - HPLC high pressure liquid chromatography - ATCC American Type Culture Collection     

Identification and mapping of a 60 bp EcoRI fragment in the Dictyostelium discoideum ribosomal DNA

We have re-examined the organization of the transcribed sequences in the ribosomal DNA repeat unit of Dictyostelium discoideum. In addition to the four EcoRI fragments previously reported, we have identified and cloned a fifth fragment which defines a small portion of the 25S ribosomal RNA. The fragment is 60 bp long and is located between the 1.5 kbp and the 3 kbp EcoRI fragments of the ribosomal DNA repeat unit.     

Organization of the nif genes in cyanobacteria in symbiotic association with Azolla and Anthoceros

The sizes of endonuclease digestion fragments of DNA from cyanobacteria in symbiotic association with Azolla caroliniana or Anthoceros punctatus, or in free-living culture, were compared by Southern hybridization using cloned nitrogenase (nif) genes from Anabaena sp. PCC 7120 as probes. The restriction fragment pattern produced by cyanobacteria isolated from A. caroliniana by culture through symbiotic association with Anthoceros differed from that of the major symbiotic cyanobacterium freshly separated from A. caroliniana. The results indicate that minor cyanobacterial symbionts occur in association with Azolla and that the dominant symbiont was not cultured in the free-living state. Both the absence of hybridization to an xisA gene probe and the mapping of restriction fragments indicated a contiguous nifHDK organization in all cells of the symbiont in association with Azolla. On the other hand, in the cultured isolate from Azolla and in Nostoc sp. 7801, the nifD and nifK genes are nominally separated by an interval of unknown length, compatible with the interruption of the nifHDK operon by a DNA element as observed in Anabaena sp. PCC 7120. In the above cultured strains, restriction fragments consistent with a contiguous nifHDK operon were also present at varying hybridization intensities, especially in Nostoc sp. 7801 grown in association with Anthoceros, presumably due to gene rearrangement in a fraction of the cells.Non-standard abbreviations bp base pairs - kb kilobase pairs - kd kilodaltons     

An estimate of the physical distance between two linked markers inHaemophilus influenzae

Using DNA clones, the physical distance between the linked genesnov andstr inHaemophilus influenzae was estimated. Although none of the cloned inserts contained both the markers, pJ1-8Str^R 13 (insert of 18·7 kb) includedstr gene at one end and part ofnov gene at the other end of the insert. By EcoRI restriction analysis and by Southern hybridization, the distance between the two EcoRI sites, cutting at which inactivates the two genes, was estimated to be 17·7 kb. A single continuous EcoRI fragment (containing 4EcoRI sites within it) carrying both the genes intact would need to be 20·4 kb in size. These estimates were confirmed independently using different clones ofnov ^r andstr ^r alleles as probes for hybridization with BamHI-digested chromosomal DNA.     

Organization and stability of endogenous xenotropic murine leukemia virus proviral DNA in mouse genomes.

In a series of blot hybridization experiments, using a xenotropic envelope probe and restriction enzymes known to cut xenotropic proviral DNA a single time (EcoRI) or not at all (HindIII), we have studied the organization and relationship of endogenous xenotropic env-related sequences in various mouse strains. Multiple copies (18 to 28) of xenotropic env-reactive fragments were found in all mouse DNAs after digestion with either HindIII or EcoRI, and the majority of fragments were of sizes compatible with their origin from full-length proviral DNA. Five HindIII and five EcoRI restriction fragments were common to all inbred mouse DNAs tested. In addition, each strain exhibited unique characteristic xenotropic env-reactive bands; these bands were remarkably stable during many years of inbreeding. The cleavage patterns characteristic of each strain were also useful for showing genealogical relatedness among the various inbred mice.     

The nucleotide sequences of two tRNAAsn genes from tobacco chloroplasts

Recombinant plasmids which contain EcoRI fragments of tobacco chloroplast DNA carrying tRNA genes were constructed. Plasmids pTC211 and pTC293 contain the base sequences for tRNAAsn in their 1.4 and 1.1 Md EcoRI fragments, respectively. These two tRNA sequences are identical and are; 5'-TCCTCAGTAGCTCAGTGGTAGAGCGGTCGGCTGTTAACCGATTGGTCGTAGGTTCGAATCCTACTTGGGGAG-3'. Each tRNAAsn gene is located at about 0.9 kb apart from the distal end of each 5S rRNA gene and is coded for by the DNA strand opposite from that of the rRNA genes.     

Epstein-Barr Virus-Specific RNA III. Mapping of DNA Encoding Viral RNA in Restringent Infection

Namalwa and Raji cells, originally obtained from a Burkitt tumor biopsy, grow as continuous cell lines in vitro and contain the Epstein-Barr virus (EBV)-related nuclear antigen EBNA (B. M. Reedman and G. Klein, Int. J. Cancer 11:499-520, 1973) and RNA homologous to at least 17 and 30% of the EBV genome, respectively (S. D. Hayward and E. Kieff, J. Virol. 18:518-525, 1976; T. Orellana and E. Kieff, J. Virol. 22:321-330, 1977). The polyribosomal and polyadenylated [poly(A)+] RNA fractions of Namalwa and Raji cells are enriched for a class of viral RNA homologous to 5 to 7% of EBV DNA (Hayward and Kieff, J. Virol. 18:518-525, 1976; Orellana and Kieff, J. Virol. 22:321-330, 1977). The objective of the experiments described in this communication was to determine the location within the map of the EBV genome (D. Given and E. Kieff, J. Virol. 28:524-542, 1978) of the DNA which encodes the viral RNA in the poly(A)+ and non-polyadenylated [poly(A)-] RNA fractions of Namalwa cells. Hybridization of labeled DNA homologous to Namalwa poly(A)+ or poly(A)- RNA to blots containing EcoRI, Hsu I, or Hsu I/EcoRI double-cut fragments of EBV (B95-8) or (W91) DNA indicated that these RNAs are encoded by DNA contained primarily in the Hsu I A/EcoRI A and Hsu I B/EcoRI A fragments and, to a lesser extent, in other fragments of the EBV genome. Hybridizations of Namalwa poly(A)+ and poly(A)- RNA in solution to denatured labeled EcoRI A or B fragments, Hsu I A, B, or D fragments, and Hsu I A/EcoRI A or Bam I S fragments and of Raji polyribosomal poly(A)+ RNA to the EcoRI A fragment indicated that (i) Namalwa poly(A)+ RNA is encoded primarily by 6 x 10(5) daltons of a 2 x 10(6)-dalton segment of DNA, Bam I S, which is tandemly reiterated, approximately 10 times, in the Hsu I A/EcoRI A fragment and is encoded to a lesser extent by DNA in the Hsu I B, EcoRI B, and Hsu I D fragments. Raji polyribosomal poly(A)+ RNA is encoded by a similar fraction of the EcoRI A fragment as that which encodes Namalwa poly(A)+ RNA. (ii) The fraction of the Bam I S fragment homologous to Namalwa poly(A)- RNA is similar to the fraction homologous to Namalwa poly(A)+ RNA. However, Namalwa poly(A)- RNA is homologous to a larger fraction of the DNA in the Hsu I B, Hsu I D, and EcoRI B fragments.     

Cleavage of DNA from 2 phages of Bacillus thuringiensis by EcoRI and HindIII endonucleases

The DNA of two Bacillus thuringiensis phages was restricted by endonucleases EcoRI and HindIII and the electrophoretic distribution of the fragments in agarose gel was studied. EcoRI was shown to restrict the DNA of phage 1-97A into 8 fragments and the DNA of phage 1-97B into 12 fragments. Restriction with HindIII results in the formation of 22 and 9 fragments for phage 1-97A and phage 1-97B, respectively. The molecular mass of the DNAs determined by summing up EcoRI restricts is 80.87 MDa for phage 1-97A and 32.45 MDa for phage 1-97B.     

Analysis of DNA associated with nucleosomes in pea chromatin

Micrococcal nuclease digestion of chromatin from ungerminated and 48 h-germinated pea embryos yields DNA fragments which are multiples of basic units of 194–195 base pairs. Extensive digestion produces a core particle of 145 base pairs. Deoxyribonuclease I gives rise to fragments which are multiples of 10 bases upon analysis on denaturing gels. These values are comparable with those found for other plant materials. These results indicate that gross changes in nucleosomal organization do not accompany the onset of germination.     

Cytological and biochemical characterization of the in situ endonuclease digestion of fixed Tenebrio molitor chromosomes

Cytological and biochemical experiments were undertaken in order to characterize the action of several restriction enzymes on fixed chromosomes of Tenebrio molitor (Coleoptera). EcoRI cuts the satellite DNA of this organism into suunit monomers of 142 bp in naked DNA and acts on fixed chromosomes cleaving and extracting these tandemly repeated sequences present in median centromeric heterochromatin. AluI, in contrast, is unable to attack the satellite sequences but does cut the main band DNA both in naked DNA and in fixed chromosomes. These enzymes therefore permit the in situ localization of satellite DNA or main band DNA in T. molitor. Other enzymes such HinfI or Sau3A do not produce longitudinal differentiation in chromosomes because of the extraction of DNA from satellite and main band DNA regions. In situ hybridization with a satellite DNA probe from T. molitor confirms that the DNA extracted from the chromosomes is the abundant and homogenous highly repeated DNA present in pericentromeric regions. These results plus the analysis of the DNA fractions retained on the slide and solubilized by the action of the restriction enzymes in situ provide evidence that: (a) as an exception to the rule EcoRI (6 bp cutter) is able to produce chromosome banding; (b) the size of the fragments produced by in situ digestion of satellite DNA with EcoRI is not a limiting factor in the extraction; (c) there is a remarkable accord between the action of EcoRI and AluI on naked DNA and on DNA in fixed chromosomes, and (d) the organization of specific chromosome regions seems to be very important in producing longitudinal differentiation on chromosomes.by E.R. Schmidt     

Cloning and characterization of a highly reiterated 5.8-kilobase pair nucleolar EcoRI DNA fragment found in Novikoff hepatoma ascites cells

The DNA of Novikoff hepatoma ascites cells was found to contain a 3.6-megadalton EcoRI restriction fragment, referred to as EcoRI fragment A (Parker et al., 1979). C0t analyses demonstrated an enrichment of fragment A sequences in Novikoff hepatoma genome relative to normal rat liver DNA. This fragment was cloned in lambda gtWES to determine its molecular structure and sequence organization. The DNA from a positive clone was labeled by nick translation and hybridized to a Southern blot of EcoRI digested Novikoff DNA. Distinct hybrids formed with the region corresponding to fragment A. The greater degree of hybridization to the nucleolar fraction suggested a nucleolar enrichment of fragment A. Fragment A has a PstI site approximately 300 base pairs from one terminus which was used to generate mono-5'-32P-labeled fragments. The larger PStI subfragment, 5500 base pairs, labeled at a single terminus, was used to evolve a restriction enzyme map. The 300 base pair fragment was partially sequenced, revealing the presence of a repetitive sequence "island", TT(GTCT)8(GAAT)5G-. C0t analysis, utilizing the purified clone as a probe, confirmed the enrichment of fragment A sequences in the tumor relative to the normal rat liver control.     

Nuclear genotype affects mitochondrial genome organization of CMS-S maize

Summary A WF9 strain of maize with the RD subtype of the S male-sterile cytoplasm (CMS-S) was converted to the inbred M825 nuclear background by recurrent backcrossing. The organization of the mitochondrial genomes of the F₁ and succeeding backcross progenies was analyzed and compared with the progenitor RD-WF9 using probes derived from the S1 and S2 mitochondrial episomes, and probes containing the genes for cytochrome c oxidase subunit I (coxI), cytochrome c oxidase subunit II (coxII) and apocytochrome b (cob). Changes in mitochondrial DNA (mtDNA) organization were observed for S1-, S2-, and coxI-homologous sequences that involve loss of homologous restriction enzyme fragments present in the RD-WF9 progenitor. With the coxI probe, the loss of certain fragments was accompanied by the appearance of a fragment not detectable in the progenitor. The changes observed indicate the effect of the nuclear genome on the differential replication of specific mitochondrial subgenomic entities.     

Physical map of the origin of defective DNA in herpes simplex virus type 1 DNA.

The origin of defective DNA (dDNA) of the Patton strain of herpes simplex virus type 1 (HSV-1) was physically mapped with BamHI in the parental DNA. The dDNA obtained from virus passaged at high multiplicities of infection was resistant to cleavage with HindIII, whereas digestion with EcoRI yielded a cluster of fragments 5.4 to 5.7 megadaltons (Mdal) in size. Cleavage with BamHI gave a cluster of fragments 2.6 to 3.2 Mdal in size, plus two homogeneous, comigrating 1-Mdal fragments. One of the latter fragments contained the single EcoRI site approximately 65 base pairs from one end. Hybridization of in vitro labeled dDNA probe to EcoRI, HindIII, BamHI, and Hpa I digests of nondefective HSV-1 DNA demonstrated that, in addition to the S-region terminal repeat, only one end of the S region was involved in the generation of this class of dDNA. Thus, the dDNA probe did not hybridize to either the S region 3.0-Mdal HindIIIN fragment or a 3.0-Mdal BamHI fragment of the adjacent 8.7-Mdal HindIIIG fragment, but did hybridize to four BamHI fragments of HindIII G (approximately 5.7 Mdal). The cluster of 2.6- to 3.2-Mdal fragments obtained with BamHI digestion of dDNA appears to represent a novel junction between the termination of dDNA adjacent to the 3.0-Mdal BamHI fragment in HindIII G and the 2.0- to 2.3-Mdal BamHI fragment terminal in HSV-1 DNA.     

The organization of genes in yeast mitochondrial DNA

Summary We have fractionated fragments of yeast mtDNA, obtained with restriction endonucleases, on poly(U)-Sephadex columns using the procedure of Flavell and Van den Berg (FEBS Letters (1975) 58, 90–93). The poly(U) forms a triple helix with (dA·dT) clusters in duplex DNA and fractionates DNA fragments on the basis of the length and number of clusters contained in them.mtDNA fragments obtained with endonucleases PstI, BamHI, HindII, HindII+III, EcoRI, HapII and HhaI were separated by poly(U)-Sephadex in three groups: fragments not retained by the column in 2M LiCl, fragments partially retained and fragments (nearly) completely bound in 2 M LiCl and only eluted by 0.1 M LiCl. The separation obtained is adequate for analytical fractionation of fragments and it can be used for the preparative isolation of firmly-bound fragments.In mtDNA digests made with endonuclease HapII, which gives about 70 separable fragments under our conditions, only about 10% of the fragments were firmly bound to poly(U)-Sephadex. This shows that the number of (dA·dT) clusters long enough to result in binding is limited in yeast mtDNA and its suggests that large fragments are bound by only one or a few clusters.Corresponding segments of the physical map of the mtDNAs from Saccharomyces carlsbergensis and Saccharomyces cerevisiae strains JS1-3D and KL14-4A were bound to the column, showing that the (dA·dT) clusters responsible for binding are conserved in the evolution of mtDNA. However, one 3,000 bp insert, only present on KL14-4A mtDNA, causes the loss of a binding site, another long insert introduces a new binding site.Fragments firmly bound to the columns are clustered in one quadrant of the physical map of these three mtDNAs. This quadrant also contains the large insertions present in KL14-4A mtDNA and absent from S. carlsbergensis mtDNA. The possible relation between (dA·dT) clusters and insertions is discussed.Abbreviation bp base pairs     

A partial map of the circular mitochondrial genome of Drosophila melanogaster. Location of EcoRI-sensitive sites and the adenine-thymine- rich region

The mitochondrial genome of Drosophila melanogaster is a circular DNA molecule of mol wt 12.35 X 10(6) daltons. A single region accounting for approx. 25% of this molecule can be reproducibly differentially denatured presumably because it is rich in adenine and thymine. We have mapped on the circular mitochondrial genome of D. melanogaster the relative positions of this adenine-thymine (A-T) rich region and the sites sensitive to cleavage by the restriction endonuclease EcoRI, using agarose gel electrophoresis and electron microscopy. Digestion of mitochondrial DNA (mtDNA) molecules to completion with EcoRI resulted in the production of four fragments, A, B, C, and D which represent (+/- SD) 58.9 +/- 1.1%, 27.5 +/- 0.8%, 8.9 +/- 0.5%, and 4.5 +/- 0.3%, of the circular genome length, respectively. Fragments produced by EcoRI digestion and circularized by incubation at 2 degrees C also fell into four distinct length groups with means (+/- SD) of 59.1 +/- 0.5%, 27.5 +/- 0.5%, 9.2 +/- 0.3%, and 4.6 +/- 0.2% of the circular genome length. From a consideration of the lengths of fragments resulting from incomplete EcoRI digestion, it was determined that the arrangement of the fragments in the circular genome was A-C-B-D. By electron microscope examination of partially denatured EcoRI fragments, the A-T- rich region was shown to be located in the A fragment closer to one end than to the other. By similar partial-denaturation studies of fragments resulting from incomplete EcoRI digestion, it was determined that, in the circular genome, of the two EcoRI sites which define the limits of the A fragment, the site between the A and D fragment lies nearest to the A-T-rich region.