The linkage mapping of cloned restriction fragment length differences in Caenorabditis elegans

Summary The genomic DNA of two closely related strains of the nematode, Caenorhabditis elegans, Bristol (N2), and Bergerac (Bo), has different restriction endonuclease sites (Emmons et al. 1979). Since these two strains interbreed, it is possible to regard the restriction fragment length differences (RFLDs) as mutant variants. The N2 and Bo pattern can be segregated and mapped using clasical genetic techniques.Utilizing a number of genetic markers existing in the N2 strain, we have constructed hybrid populations homozygous for either Bristol or Bergerac over a given chromosomal region with random Bristol-Bergerac composition for the remainder of the genome. Genomic restriction digests from these hybrid populations were probed with random cloned fragments of Bristol DNA. In this way, fragments were mapped to genetically well characterized regions of the C. elegans genome. 27 probes which hybridize to a total of 310 Kb of DNA were found to exhibit six restriction fragment differences. Four of these differences have been mapped, providing probes for four different genomic regions. We have combined classical genetics and recombinant DNA technology to construct linkage maps of cloned DNA fragments using restriction fragment length differences. We are pursuing this approach in order to advance the knowledge of the genetic organization of C. elegans and to provide a means of cloning genes in an organism which provides an experimental model for the study of many biological systems. It is hoped that this approach will also provide a practical solution to some difficult problems in nematode strain identification. Furthermore, the characterization of the families of transposable elements responsible for generating many of the RFLDs will undoubtedly contribute to the understanding of the biological significance of these elements.     

Cloning and expression of Bacillus subtilis spore genes

Summary Two spore genes, spoOB and spoIIG have been cloned from the B. subtilis genome library, constructed by ligating Sau3A partially digested DNA to the dephosphorylated pHV33 plasmid vector at its BamH1 site.An hybrid plasmid pGsOB2, carrying a 1.7 Kb insert of B. subtilis DNA amplifiable in E. coli was cloned. This recombinant plasmid was capable of transforming the appropriate B. subtilis Rec⁺ and Rec^- recipients to Spo⁺ at very high efficiency. The pGsOB2 was further subcloned and four hybrid plasmids, pGsOB8, pGsOB9, pGsOB10 and pGsOB11 were selected and their restriction enzyme maps established. The four subcloned hybrid plasmids retained their entire transforming activity in both Rec⁺ and Rec^- recipients although two of them carry the insert in an inverse orientation, indicating thus, that the spoOB gene in these plasmids is being transcribed by the B. subtilis RNA polymerase using an internal promotor of the cloned DNA fragment. The adjacent genes spoIVF and pheA, mapped respectively to the right and left of the spoOB locus, that normally show 90% cotransformation, are absent on the cloned DNA fragments. The cloned hybrid plasmids have been expressed in E. coli minicells and it was shown that the spoOB locus encoded a polypeptide of 24 K.We have also cloned the spoIIG gene in two hybrid plasmids, pGsIIG24 and pGsIIG26, carrying respectively inserts of 2 and 3 Kb. From the transforming activity and the endonuclease cleavage maps it was shown that these two hybrid plasmids do not carry the entire spoIIG locus. The use of these plasmids for further cloning of this gene is discussed.     

Cloning and expression of a glucoamylase gene from Lactobacillus amylovorus ATCC 33621 in Escherichia coli

Summary The glucoamylase gene from Lactobacillus amylovorus was cloned and expressed in Escherichia coli. A genomic DNA library from Lactobacillus amylovorus was prepared by partially digesting genomic DNA with EcoRI and ligating random fragments to the EcoRI digested cloning vector, pZErO-1.1. Three E. coli transformants expressing glucoamylase were identified using a probe prepared from the STA2 glucoamylase gene from Saccharomyces cerevisiae var. diastaticus. The physical maps of the recombinant plasmids were constructed. These plasmids contained inserts of about 5.2 Kb, 5.9 Kb and 6.4 Kb respectively. Temperature and pH optima of 45°C and 6.0, respectively, were obtained for both recombinant and purified wild type glucoamylases. Also, the enzymes were found to be thermolabile at temperatures above 50°C.     

Cloning of Petunia hybrida chloroplast DNA sequences capable of autonomous replication in yeast

Summary Sequences from Petunia hybrida chloroplast DNA which have the property to promote autonomous replication in Saccharomyces cerevisiae were cloned in vector YIp5. Seven cloned chloroplast DNA fragments are localized at one of two different sites on the chloroplast genome. One site, arsA was mapped on a 1.8 Kb fragment at position 27.0–28.8 Kb on the P. hybrida chloroplast genome. The plasmids containing this arsA are stable both in yeast and E. coli. The other site, arsB, was shown to be very unstable and is located either in the small single copy region close to the inverted repeat or just in the inverted repeat. The functioning of these sequences as a possible origin of replication in vivo is discussed.     

Expression of a β-galactosidase gene from Lactobacillus sake in Escherichia coli

Summary A -galactosidase gene from Lactobacillus sake coding for lactose hydrolysis was cloned and expressed in Escherichia coli. Chromosomal DNA from L. sake was partially digested with the restriction enzyme Sau3AI, and the 3–6 Kb fragment was ligated to the cloning vector pSP72 digested with BamHI. One E. coli transformant expressing -galactosidase was isolated on X-gal plates. It contained a plasmid with an insertion of approx. 4 Kb. The restriction map of the recombinant plasmid was constructed. The characteristics of the recombinant -galactosidase were compared with those of the wild type. The optima pH and temperature for both enzymes was 6.5 and 50°C, respectively. Stability of the enzymes at different temperatures and activity on lactose were determined.     

Cloning of mitochondrial DNA from the petite negative yeast Schizosaccharomyces pombe in the bacterial plasmid pBR322

Summary The entire mitochondrial (mt) genome of the yeast Schizosaccharomyces pombe (S. pombe) was cloned in the BamHI site of the Escherichia coli plasmid pBR322. Three lines of evidence demonstrate that the complete mtDNA molecule was amplified without rearrangement or partial loss. First, restriction of the hybrid plasmid with BamHI led to the recovery of two fragments corresponding to the linearized plasmid and the BamHI-cut mtDNA. Second, restriction of cloned and native mtDNA with HindIII revealed identical fragments. Third, mitochondrial ribosomal RNA hybridized to the same HindIII fragments from cloned mtDNA and from mtDNA isolated from mitochondria.     

The large endogenous plasmid of Anacystis nidulans: Mapping, cloning and localization of the origin of replication

Summary Anacystis nidulans contains two cryptic plasmids of 8.0 (pANS) and 48.5 (pANL) kilobasepairs (kbp). A clone bank of the large plasmid pANL consisting of 7 Bam HI fragments has been established. The cloned fragments were used as radioactive probes to Bam HI, Sal I, Hind III and Eco R1 digests of pANL in blot hybridization experiments to verify the clones and map the restriction fragments. Further size characterization of the physical map was done by restriction analysis of the cloned fragments. The origin of replication has been located in the largest Bam HI fragment of the large plasmid.     

Identification and molecular analysis of the cuticle protein genes of Dacus oleae

A 11.9 Kb DNA segment of the Dacus oleae genome that contains three cuticle protein genes has been cloned and characterized. These three genes are clustered within 8.04 Kb of DNA; the restriction sites for eight enzymes and the organization of the cuticle genes in this 11.9 Kb cloned fragment were determined. Using this clone as a radioactive probe, it was shown that the three cuticle genes are expressed as poly(A) RNA in the epidermis of late third instar larvae but are not abundantly expressed in other tissues.     

Cloning part of the region encoding biosynthetic enzymes for surface antigen (O-antigen) of Salmonella typhimurium

Summary The rfb gene cluster of Salmonella typhimurium encodes the enzymes required for the biosynthesis of the O-Antigen. A part of it has been cloned in plasmid vectors pBR322 and pUC9 using an adjacent, previously cloned, part of the his operon (Barnes 1981) as a molecular probe for the first clone. A detailed restriction enzyme map of 7.57 kb of rfb DNA is presented and the approximate locations of two of the genes, rfbK and rfbM have been defined.     

Localisation of identical organophosphorus pesticide degrading (opd) genes on genetically dissimilar indigenous plasmids of soil bacteria: PCR amplification,cloning and sequencing of opd gene from Flavobacterium balustinum

Plasmid borne organophosphorus pesticide degrading (opd) gene of Flavobacterium balustinum has been amplified using polymerase chain reaction (PCR) and the resulting PCR product (1.25 Kb) was cloned in pUC18. Further, a detailed restriction map was determined to PCR product and subcloned as overlapping restriction fragments. The nucleotide sequence was determined for all subclones to obtain complete sequence of PCR amplified fragment. The sequence showed 98% similarity to opd genes cloned from other soil bacteria isolated from diversified geographical regions. The protein sequence predicted from the nucleotide sequence was almost identical to parathion hydrolase, a triesterase involved in hydrolysis of triester bond found in variety of op-pesticides. The signal sequence of parathion hydrolase contained recently discovered twin arginine transport (tat) motif. It appears that tat motif plays a critical role in membrane targeting of parathion hydrolase.     

Histone genes in macronuclear DNA of the ciliate Stylonychia mytilus

DNA in the macronucleus of Stylonychia mytilus exists as discrete gene-sized fragments which are derived from micronuclear DNA through a series of well-defined developmental events. It has been proposed that each of the DNA fragments might represent a gene and its controlling elements. We have investigated this possibility using genes which code for the five histone proteins. Macronuclear DNA fragments were fractionated according to size by agarose gel electrophoresis, the fragments transferred to nitrocellulose filters using the technique of Southern, and the filter-bound DNA hybridized with labeled cloned histone genes of the sea urchin, Psammechinus miliaris. Results indicate, first, that sequences homologous to the five individual histone gene probes are present in discrete macronuclear fragments which appear as bands in the gel hybridization assay. Secondly, for each of the five individual histone gene probes the homologous DNA fragments are several in number, ranging in size from 7.6 Kb (Kilo base pairs) to 0.73 Kb. For example, the largest of six detected fragments hybridizing to the H3 gene probe contains approximately 10 times the amount of DNA required to code for a Stylonychia H3 histone. The smallest detected fragment hybridizing to the H3 probe contains enough DNA to code for approximately two copies of the histone. Finally, in general, no two histone gene probes hybridized to the same macronuclear DNA fragment. This result indicates that genes coding for the five histones in Stylonychia are not located together on the same macronuclear DNA fragments and implies that the five functionally related genes would not be transcribed together as a polycistronic unit.     

Isolation of a recombinant lambda phage carrying nusA and surrounding region of the Escherichia coli K-12 chromosome

Summary A recombinant bacteriophage lambda, argG-6, has been isolated which carries the argG gene and neighbouring loci on an EcoRI-generated 15.5 Kb DNA fragment from the Escherichia coli chromosome. The locations of the argG, nusA and pnp genes on the 15.5 Kb DNA fragment have been determined. In the case of nusA, a Tn5 insertion and sub-cloning of restriction fragments were used to locate the gene. The gene products of nusA and pnp have been identified on one- and two-dimensional polyacrylamide gels. The clockwise gene order was found to be argG-nusA-pnp.     

Mitochondrial DNA of the basidiomycete Polyporus ciliatus

Summary Mitochondrial (mt) DNA of the white rot fungus Polyporus ciliatus was isolated and characterized. As a result of detailed restriction enzyme analysis, a physical map was established showing that this circular DNA has a molecular weight of 88.2 kb. By heterologous cross hybridization the sites of three mt genes were recognized. By nonselective cloning of mt DNA fragments in Saccharomyces cerevisiae, an autonomously replicating sequence (ars) was identified which has potential application in the development of a prokaryotic/eukaryotic shuttle vector.     

The genome of Bacillus subtilis phage SPP1: the arrangement of restriction endonuclease generated fragments.

Summary SPP1 DNA was cleaved by the restriction endonucleases, BglI, BglII, EcoRI, KpnI, SmaI, and SalI. The molecular weights of the DNA fragments obtained by single enzyme digestion or by consecutive digestion with two enzymes were determined by electron microscopic measurements of contour length and by gel electrophoresis. The major fragments from the six digests could be ordered to give a consistent restriction map of SPP1. The electropherograms of several digests indicated that certain fragments occurred in less than stoichiometric amounts or were heterogeneous in size. Such bands carried a major part of radioactivity, when SPP1 DNA was terminally labelled with P³² prior to degradation by restriction enzymes. These results, and studies of the effect of exonuclease III treatment on restriction enzyme patterns define the terminal restriction fragments. All data obaained support the conclusion drawn in the preceding paper (Morelli et al., 1978b) that the SPP1 genome is terminally redundant and partially circularly permuted.Part of this work is from the doctoral dissertations to be submitted to Stanford University¹ and the Freie Universität Berlin²     

High-resolution mapping of genetic loci of Anabaena PCC 7120 required for photosynthesis and nitrogen fixation

A physical map of the Anabaena genome permitted the localization of its genes to chromosomal fragments generated by rarely cutting restriction endonucleases and separated by pulsed-field gel electrophoresis. We introduce a novel means of mapping more precisely to c. 20 kb by use of rare restriction sites within vectors bearing cloned sequences that undergo homologous recombination with the genome. We thereby localize and orient genes encoding principal photosynthetic pigments. The relative spacing of loci within a single restriction fragment was determined with even higher resolution, as illustrated for genes required for heterocyst development and nitrogen fixation that were marked with transposons. Small, newly visualized restriction fragments of the chromosome were also mapped.     

Physical characterization of the genome of the Myxococcus xanthus bacteriophage MX-8

Summary We have constructed a restriction map for the genome of bacteriophage MX-8 from Myxococcus xanthus using the enzymes PvuII, MboI, and EcoRI. The phage genome size, as determined by restriction analysis, is 51.7±0.6 Kb. Double digestions, redigestions of isolated fragments, and crossed-contact hybridization of partial digestion products show that the restriction map is circular. Restriction analysis and Southern hybridization show that the phage DNA molecules are packaged sequentially from a concatemer starting from a specific site which we have mapped. The DNA molecules have an average terminal redundancy of approximately 8% and are circularly permuted over at least 40% of the genome.     

Cloning of two protease genes fromRhodocyclus gelatinosa APR 3-2 and their expression inEscherichia coli

Two different protease genes were cloned fromRhodocyclus gelatinosa APR 3-2 inEscherichia coli HB 101/ with pBR329 or its derivatives. The recombinant plasmids designated as pRP100 and pRP300 contained 11.2 and 10.6 kb DNA fragments, respectively. The differences of both plasmids in restriction enzyme maps indicate that these plasmids contained different protease genes. DNA fragments coding for protease, 6.4 kb and 4.5 kb from pRP100 and pRP300, were subcloned into pRP329 and designated as pRP101 and pRP301, respectively. The two cloned proteases were excreted in culture medium ofE. coli, and ß-lactamase ofE. coli, which was originally localized in periplasmic space, was also excreted in the medium.     

Kinetoplast DNA in the insect trypanosomes Crithidia luciliae and Crithidia fasciculata: II. Sequence evolution of the minicircles

Minicircle sequence evolution has been studied by comparison of the minicircles from Crithidia fasciculata and C. luciliae (C. fasciculata, var. luciliae) by restriction enzyme analysis and hybridization experiments. In contrast to the maxicircle sequence, the minicircle sequence of these trypanosomes evolves very rapidly. No conservation of restriction fragments has been observed and cross-hybridization of minicircles, minicircle fragments, and total kDNA is relatively weak. We conclude that no fragment larger than 550 bp is perfectly conserved between all minicircles of the two trypanosomes. Alterations in the minicircle fragment patterns of our stocks of trypanosomes were even apparent in a cultivation period of 1.5 to 2 years. The alterations suggest a random drift of the sequence which supports a noncodogenic function for the minicircles. Double restriction enzyme digestion experiments show that primary fragments are homogeneous with respect to cleavage by the second enzyme. This suggests that sequence rearrangements, rather than point mutations are the basis of the minicircle sequence heterogeneity.     

A repetitive and apparently transposable DNA sequence in Dictyostelium discoideum associated with developmentally regulated RNAs.

Dictyostelium discoideum AX3 genomic DNA contains about 40 copies of a 4.5 Kb sequence. Each has the same restriction map, suggesting that they are all very similar. The sequences are scattered throughout the genome, and each of the six representative copies we cloned have different flanking sequences. Partial fragments of the 4.5 Kb sequence also are scattered throughout the genome. The DNA sequences flanking the 4.5 Kb repetitive sequences are different in different strains of Dictyostelium suggesting that the 4.5 Kb sequence is a transposable element. Each sub-region of this 4.5 Kb sequence is associated with a large number of mRNAs, all of which are developmentally regulated, but whose function is not known.