Cornelis P. Hollenberg

Non xenopus-like DNA sequence organization in the Chironomus tentans genome

SummaryWe have examined the sequence organization of Chironomus tentans DNA by means of optical and hydroxyapatite renaturation kinetics of total DNA fragment sizes of 0.36, 2.6 and 13.5 kilobases (kb) as well as isolated middle repeat DNA at sizes of 0.36 and 13.5 kb. 90% of the DNA renatured as unique sequences of a genome of 0.20 pg with the balance of DNA renaturing as middle repetitive sequences present on average 90 times per haploid genome. At a DNA fragment length of 13.5 kb, 35% of the DNA was trapped on the hydroxyapatite as middle repetitive fraction. We concluded C. tentans DNA to have a mean repeat length of about 4.3 kb distributed through out at least 35% of the genome with an inter repeat spacing of at least 13.5 kb but possibly being distributed throughout the whole genome with an inter repeat spacing of 36 kb. This shows C. tentans DNA organization not to follow the almost ubiquitous Xenopus model but to be similar to the organization of Drosophila melanogaster DNA.

Synthesis of high molecular weight polypeptides in Escherichia coli minicells directed by cloned Saccharomyces cerevisiae 2-μm DNA

The minicell-producing Escherichia coli strain P 678-54 was transformed with a series of defined PTY chimeric plasmids consisting of yeast 2-micron DNA and E. coli plasmid pCR1. In minicells the integrated 2-micron DNA from yeast directed specifically the synthesis of six polypeptides with apparent molecular weights of 15,000, 17,500, 20,000, 22,000, 37,000, AND 48,000. The specificity of five other polypeptides, which cover a molecular weight range of 19,000 to 28,000, has not yet been established with certainty. Neither the orientation of the integrated DNA, nor the inversion which distinguishes the two structural forms of 2-micron DNA affected the polypeptides synthesized. However, integration at a given EcoRI site appeared to be correlated with the absence of one particular polypeptide band; this suggests that at least one of these sites is located in an expressed region of the DNA.

The organization of the ribosomal RNA genes of Chironomus tentans and some closely related species.

Southern gel analysis of total DNA from Chironomus tentans showed that the rRNA genes (rDNA) are homogeneous in structure. After cloning in Escherichia coli plasmid pBR313, the rDNA organisation was further studied by restriction fragment analysis and R-loop mapping. No heterogeneity could be detected by heteroduplex analysis of six different cloned rRNA cistrons. R-loop sizes of 1.69 and 3.63 kilobases (kb) were measured for the 18S and 28S rRNA coding sequences. The two spacers are 0.75 and 1.77 kb long. Southern gel analysis showed also a homogeneous rDNA structure for a Canadian population of C. tentans and C. pallidivittatus. The same technique indicated, however, that the rDNA of two other closely related species of C. thummi and C. melanotus is heterogeneous in structure. A possible correlation between this heterogeneity and the presence of heterochromatin in these species is discussed.

Mapping of regions on cloned saccharomyces cerevisiae 2-?m DNA coding for polypeptides synthesized in Escherichia coli minicells

SummarySaccharomyces cerevisiae 2-μm DNA and some of its restriction fragments were integrated in vector pCR1, pBR313 or pBR322 and their expression in Escherichia coli P678-54 minicells was analyzed. 2-μm DNA inserted at the EcoRI site of pCR1 or pBR313 and at the PstI site of pBR322, promoted the synthesis of polypeptides of 48,000, 37,000, 35,000 and 19,000 daltons. The DNA regions coding for these polypeptides were mapped on the 2-μm DNA molecule by insertion of single EcoRI or HindIII restriction fragments and comparison of the polypeptides produced. For the synthesis of the 37,000 dalton polypeptide, intact sites RIB and H3 were required. The disappearance of the 37,000 dalton polypeptide on interruption of one of these sites by insertion of the vector, was correlated with the appearance of a polypeptide of 22,000 or 23,500 daltons repectively. The DNA sequence coding for the 37,000 dalton polypeptide, therefore, has to be located in the S-loop region close to or overlapping with the sites RIB and H3. Assuming that the 22,000 and the 23,500 dalton polypeptides are truncated forms of the 37,000 dalton polypeptide, the last polypeptide can be exactly mapped. The polypeptide of 48,000 daltons was mapped to that half of the L-loop segment containing the sites H1 and H2. If, however, HindIII fragment H1-H2 was expressed, the 48,000 dalton polypeptide was lost and concomitantly a 43,000 dalton polypeptide appeared. We assume that this polypeptide results from early termination of the polypeptide of 48,000 daltons. The 35,000 and 19,000 dalton polypeptides were mapped to the S-loop region.The integrated inverted repeat sequence of yeast 2-μm DNA did not induce any detectable insert-specific polypeptide synthesis.

A structural analysis of Balbiani ring DNA sequences in Chironomus tentans

The Balbiani rings in the salivary gland polytene chromosomes of Chironomus tentans include the most active structural genes in this organ. Two of them (BR1 and BR2) contain repetitive sequences and are transcribed into giant RNA molecules. On Southern blots of restriction digests, we have identified fragments of genomic DNA which contain BR sequences. One of these fragments with a length of about 150 bp has been cloned and shown to hybridize preferentially to the BR1 transcription unit. Determination of its nucleotide sequence revealed several recognition sites for restriction enzymes which cleave the giant BR gene(s) into small pieces of approximately 240 bp. It is concluded that the cloned fragment represents part of the basic 240 bp repeat unit of a BR1 gene. Data obtained from partial restriction digests using the cloned DNA segment as a probe indicate that probably the entire BR1 gene comprises tandem repeats of 240 bp. Evidence is presented that the cloned BR1 sequence significantly cross-hybridizes to BR2 and to a lesser extent to BR6. BR2 sequences are present on a MboI fragment of 40 kb and seem to be organized in a very similar way as found for the BR1 gene.

Cloning and characterization of the ribosomal RNA genes of Rhynchosciara americana.

The ribosomal RNA genes (rDNA) of Rhynchosciara americana were analysed using Southern transfers of DNA cleaved with EcoRI, HindIII, BamHI and PstI. The results show that the rDNA is heterogeneous in structure. Following digestion with EcoRI and hybridization to rRNA three bands corresponding to fragments of 9.5, 7.5 and 5.5 kilobases (kb) were detected. Recombinants containing EcoRI fragments of R. americana DNA were prepared using the vector λgtλB. Three different recombinants (λgtRa1, λgtRa23 and λgtRa5) were isolated containing the rDNA fragments of 9.5, 7.5 and 5.5 kb, respectively. These fragments were transferred to pBR325 and analysed with restriction enzymes and Southern hybridization with 28 S and 18 S rRNA. The λgt recombinants were further analysed by R-loop mapping. The data show that the rDNA occurs in two different repeating gene units. A shorter repeat of 9.5 kb and a longer repeat of 13 kb, in which the 28 S rRNA coding sequence contains an insertion of 3.5 kb.

Saccharomyces cerevisiae 2-μm DNA

SummaryThe non-tandem inverted duplication in the 2-μm DNA of Saccharomyces cerevisiae has a length of 0.19 μm and is located asymmetrically along the molecule. The majority of the dumb-bell structures that are formed upon denaturation and selfannealing of the 2-μm monomer consists of the renatured inverted duplication sequences as double stranded stem and two single stranded loops of 0.67 μm±0.06 μm (S-loop) and 0.86 μm±0.05 μm (L-loop) length. Two additional size classes which comprised 5–10% of the measured molecules had contour lengths of around 1.7 μm and 2.1 μm. The smaller dumb-bells contained two S-loops and the larger dumb-bells contained two L-loops as was shown by heteroduplex mapping with an HindIII fragment from the L-loop. Two models which assume illegitimate or site specific recombination, are presented to explain the generation of double S-loop and double L-loop molecules. At least part of the 4-μm and 6-μ circular molecules present in the yeast supercoiled DNA fraction are shown to be dimers and trimers of 2-μm monomers, but often with inverted loop segments most probably due to intramolecular recombination between sequences of the inverted duplication.

Inheritance of multiple drug resistance in Saccharomyces cerevisiae: linkage to leu1 and analyses of 2 micron DNA in partial revertants.

SummaryThe inheritance and phenotype of multiple drug resistance in independent multiple drug resistant mutants, two isolated in this laboratory (GR359 and 2–20), and two (DRI 9/T7 and DRI 9/T8) reported by Guerineau et al. (Biochem. Biophys. Res. Commun. 61,462), was investigated. Comparison of resistance to 12 selected drugs showed that the resistance phenotypes of all mutants were similar, although some differences in levels of resistance of each mutant was observed with certain drugs. Mapping of the resistance loci in GR359 and 2–20 revealed tight linkage of both resistance genes to the centromere linked gene leu1. 2 μm DNA was analysed by hybridization of 2 μm RNA to EcoRI fragments of a total DNA extract. Eight partial revertants of 2–20, which had been chosen as having a phenotype similar to the 2 μm DNA deficient [ciro] isolate DRI 9/t7, revealed the presence of 2 μm DNA. The lack of detectable 2 μm DNA in DRI 9/t7 was confirmed.

Mapping of Escherichia coli RNA polymerase binding sites on 2-μm DNA from Saccharomyces cerevisiae: Heterogeneity within the inverted duplication and evidence for an eukaryotic invertible DNA sequence

Abstract The 2-μm DNA plasmids from Saccharomyces cerevisiae strain H1 and strain HQ/5C were analyzed by electron microscopy for the presence of Escherichia coli RNA polymerase binding sites. On native 2-μm DNA isolated from strain HQ/5C five RNA polymerase binding sites were detected. One further site was mapped on cloned 2-μm DNA type 23 from S. cerevisiae strain H1. This additional site is located at a distance of 2.15 kilobases from Eco RI site B inside one of the inverted duplication (id) sequences. No such binding site could be detected in the other id sequence of the type 23 molecule, thus indicating that the two id sequences of strain H1 differ in at least one short region. The location of the id sequence carrying the RNA polymerase binding site was analyzed in native 2-μm DNA isolated from strain H1 and found to be present on Hin dIII fragment 2 and absent from Hin dIII fragment 5. This indicates that at least a part of the id sequences has a fixed position with respect to the unique S segment and further suggests a site specific recombination mechanism for the inversion of one of the unique segments. As a control for the specificity of RNA polymerase binding, we have mapped binding sites on vectors pBR313 and pBR322. The location of the E. coli RNA polymerase binding sites on 2-μm DNA is discussed in relation to the DNA regions expressed in E. coli minicells.

Recombination: Recombinant DNA Research, Techniques and Results

During the last few years, a biological method for the isolation of specific DNA fragments has been developed, and its application is continuously being improved and extended. This technique of DNA cloning makes use of bacteria and bacterial plasmids to segregate and propagate foreign DNA and allows the isolation of almost any gene or DNA fragment which can be identified outside the organism of origin. The in vitro recombination of unselected DNA fragments with a bacterial vector DNA constitutes a crucial step in all DNA cloning procedures, and has played an important role in the development of molecular cloning. The method can be applied to all biological problems studied at the DNA level and with its broad applicability, it can be compared with a technique like molecular hybridization.