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PubMed1978

Mapping the spliced and unspliced late lytic SV40 RNAs.

The sizes and map positions of the major late lytic SV40 cytoplasmic mRNAs and the abundant nuclear RNA species have been determined by the technique of Berk and Sharp (1977, 1978). From these experiments, the coding sequences (bodies) of the 16S and 19S late cytoplasmic SV40 RNAs have been located at 0.935-0.17 and 0.765-0.17 map units, respectively. The cytoplasmic 16S RNA molecules contain a leader sequence of approximately 210 nucleotides, corresponding to SV40 map positions 0.72-0.76 units, spliced to the coding sequences. In a population of the late 19S RNA molecules, there are several different leader segments, each spliced to the same coding sequences. The size of these 19S leader RNA segments was estimated to be 50-70, 100-120 and 200-210 nucleotides in length. The 5' ends of the 19S leader RNA segments were located at 0.72, 0.71, 0.695 and 0.69 map units. An analysis of the nuclear viral RNAs has provided insight into the biogenesis of the cytoplasmic messages. Poly (A)-containing nuclear RNA has a number of species in addition to those found in the cytoplasm. The 3' ends of the poly (A)-containing RNAs map at 0.17 SV40 units. The 5' ends of the more abundant nuclear molecules map approximately at 0.72, 0.70, 0.67, 0.64 and 0.59 units. Since these nuclear SV40 RNA molecules are both colinear with the viral DNA and larger than the cytoplasmic nRNAs, they may represent intermediates in a stepwise processing system. Alternatively, the variation in 5' ends of nuclear SV40 transcripts may represent a number of separate initiation sites for transcription. The presence of the intervening RNA sequences (between the leader and the coding sequences of the mature mRNAs) in these nuclear RNA molecules suggests that the synthesis of "spliced" SV40 RNA involves the direct transcription of the DNA sequences and the subsequent splicing out of the intervening seqment of RNA. Evaluation of the more abundant nonpolyadenylated nuclear RNA molecules showed that they have the same 5' ends as the poly (A)-containing nuclear RNAs. The 3' ends of the nonpolyadenylated RNA molecules map heterogeneously in a broad region extending beyond 0.28 map units. The presence of these long nuclear viral transcripts suggests that transcription of late SV40 RNA does not terminate at 0.17 map units. The location of poly (A) in mature cytoplasmic viral RNA at 0.17 map units suggest that poly (A) addition to RNA molecules may occur by a specific cleavage of the longer transcripts. Based on these analyses, we propose that the longer nonpolyadenylated viral RNA molecules in the nuclei of SV40-infected cells may represent the primary transcripts. While their 5' termini are being processed, the specific addition of poly (A) at 0.17 map units takes place. The polyadenylation of RNA is followed by splicing events to generate the cytoplasmic forms of SV40 mRNA.

Base Sequence↗
PubMed1979

Physical map of chromosomal nitrogen fixation (nif) genes of Klebsiella pneumoniae.

We describe a method for the rapid determination of the physical location of mutations caused by insertion of transposable elements. We used this method to construct a detailed physical map of the nitrogen fixation (nif) gene cluster of Klebsiella pneumoniae and to correlate it with the genetic map. Total cellular DNA was isolated from individual strains, each carrying an insertion in 1 of 15 different nif genes. The DNA was digested with a restriction endonuclease, fractionated by agarose gel electrophoresis, denatured, and blotted onto nitrocellulose filter paper. The DNA on the filters was hybridized with (32)P-labeled DNA fragments derived from amplifiable plasmids carrying cloned nif DNA fragments from K. pneumoniae. Altered hybridization patterns caused by insertions into nif genes allowed us to map nif mutations with respect to the previously mapped cleavage sites for various restriction endonucleases. We have used the same method to map the end points of nif deletions. Using this procedure, we assigned physical locations on the K. pneumoniae chromosome to 86 nif insertion mutations and 13 nif deletion end points. This mapping procedure provides a convenient alternative to deletion mapping as a definitive method for mapping insertion mutations within a gene or for ordering genes within a gene cluster. This procedure will be especially useful for mapping mutations conferring phenotypes that are difficult to monitor and for mapping mutations in bacterial species in which techniques for conducting deletion mapping have not been devised.

Bacteriophages↗
PubMed1979

Electron microscopic mapping of RNA transcribed from the late region of polyoma virus DNA.

The polyoma virus (Py) RNA species transcribed from the L DNA strand of the "late" region of the Py genome in Py-infected mouse cells have been mapped by hybridization with specific fragments of Py DNA followed by electron microscopic visualization of the hybrids. Total cellular polyadenylated Py-specific RNA molecules having an S value in the range of 16S to 20S were purified by oligodeoxythymidylic acidcellulose column chromatography, preparative hybridization with Py DNA, and sucrose gradient centrifugation. Cytoplasmic Py-specific RNA was similarily purified, except that it was not fractionated by sucrose gradient centrifugation. Hybrids of these RNA molecules and Py DNA fragments were spread for electron microscopy by either the cytochrome c technique or the bacteriophage T4 gene 32 protein method. The polyadenylic acid at the 3'-end of the RNA in the hybrids was identified by labeling with simian virus 40 DNA circles to which polybromodeoxyuridylic acid tails had been covalently attached. These experiments revealed the presence of three L DNA strand transcripts in both RNA preparations. Two of these RNA molecules were found to be spliced from chains transcribed from two noncontiguous parts of the late region. The third molecule either is a continuous transcript of the entire late region or contains a splicing feature which is too small to be reliably observed by the electron microscope methods used. The 5'-ends of the three RNA species map within a region extending from 68 to 70 map units on the Py restriction endonuclease map. Each of the two spliced molecules contains a 5'-terminal leader sequence transcribed from a DNA segment with an estimated length of 60 to 110 nuvleotides. The 3'-ends of the leaders map at 66.7 +/- 1.0 and 66.4 +/- 0.50 map units. In these molecules the 5'-ends of the other part (the main body) map at 59.4 +/- 0.90 and 49.4 +/- 2.0 map units, respectively. The 3'-termini of all three RNA species map at 24 to 25 map units.

DNA, Viral↗
PubMed1976

Prophage map of converting corynebacteriophage beta.

A prophage map for corynebacteriophage beta consisting of seven markers has been constructed and compared with the vegetative map. The mapping system utilizes heteroimmune double lysogens and capitalizes on the fact that these double lysogens are very unstable and throw off monolysogenic segregants. The prophage map, produced by characterizing the recombinant phage in these monolysogenic segregants, appears to be a cyclic permutation of the vegetative map with the gene for toxin at one end of the prophage map and the gene for phage immunity at the other. This permutation is in accord with the Campbell model for insertion of lambda phage if a site between the toxin and immunity genes in the vegetative map is designated as the phage attachment site. The position of the gene for toxin in the prophage map suggests that converting phages may have originated as specialized transducing phages for this gene.

Bacteriophages↗
PubMed1979

The orientation of the visuotectal map in Xenopus: developmental aspects.

Rotations and translocations of the eye anlage were performed in Xenopus embryos of stages ranging from 21/22 to 30. Some of the operations involved grafting wild-type eye anlagen into albino host orbits. Operations were performed under a variety of operating media and conditions. In later larval life, or after metamorphosis, the visuotectal maps from the operated eyes were recorded electrophysiologically. Results fell into two classes. In the majority, the orientation of the visuotopic map corresponded to the orientation of the eye at the time of recording. In the minority the visuotopic maps were 'compound', consisting of two parts each with its own independent orientation. The organization of the compound maps was such that one component was oriented in correspondence with the orientation of the eye, while the other component was normally oriented. Histological analysis and observations on genetically marked grafts indicated that the component parts of the compound eye were of dual cellular origin. The component giving the rotated (or translocated) map belonged to the originally operated eye tissue; whereas the component giving the normally oriented map was derived from newly grown eye tissue coming from the optic stalk. In no case was a normally oriented map obtained from a rotated or translocated eye. The results are discussed in relation to mechanisms proposed to account for the determination of map-related retinal specificity.

Animals↗
PubMed1976

Comparative mapping using somatic cell hybrids.

Comparative mapping, or ascertaining the gene linkage relationships between different species, is rapidly developing. This is possible because new techniques in chromosome identification and somatic cell hybridization, such as the generation of hybrids preferentially segregating chromosomes of any desired species including rodents, and the development of gene transfer techniques have yielded new information about the human and rodent gene maps. In addition, the discovery and characterization of mouse subspecies has generated new mouse sexual genetic linkage data. The following picture is emerging. Several X-linked genes in man are X-linked in all mammalian species tested. The linkage relationships of several tightly linked genes, less than 1 map unit apart, are also conserved in all mammalian species tested. Ape autosomal genes are assigned to ape chromosomes homologous to their human counterparts indicating extensive conservation in the 12 million years (MYR) of evolution from apes to man. Similarly, mouse and rat, 10 MYR apart in evolution, have several large autosomal synteny groups conserved. In comparing the mouse and human gene maps we find that human genes assigned to different arms of the same human chromosome are unlinked in the mouse; mouse genes large map distances (20 to 45 cM) apart are very likely to be unlinked in the human. However, several autosomal synteny groups 10 to 20 cM apart, including the Pgd, Eno-1, Pgm-1 group on human chromosome arm 1p, are conserved in mice and man. This suggests that homology mapping, the superimposition of one species gene map on the homologous conserved portion of another species genome may be possible, and that ancestral autosomal synteny groups should be detectable.

Animals↗
PubMed1977

R loop mapping of the 18S and 28S sequences in the long and short repeating units of Drosophila melanogaster rDNA.

Cleavage of D. melanogaster rDNA with the Eco R1 restriction endonuclease reveals two major classes of repeating units: a long class of 17 kilobases (kb) and a short class of 11.4 kb. R loop mapping has been used to determine the topography of the sequences corresponding to the 18S and 28S rRNAs in both classes, including a cloned member (Dm103) of the long class. This mapping procedure derives from a novel reaction that we discovered between single-stranded RNA and homologous regions in duplex DNA molecules. In high formamide and at an elevated temperature, the RNA pairs with one of the two DNA strands in the region of homology to form an R loop in which one element is an RNA/DNA duplex and the other is single-stranded DNA (Figure 1). Mapping is accomplished by visualization of R loops in the electron microscope. The R loop map of Dm103 parallels that determined independently by Glover and Hogness (1977) from an analysis of its restriction fragments. Both maps indicate that the 28S rDNA in this cloned unit is divided into two blocks by a 5 kb insertion segment. R loop mapping of a population of long units obtained directly from the rDNA (that is, without cloning) has demonstrated that this interruption of the sequence coding for the 28S rRNA is a characteristic of the long class. By contrast, the 28S rDNA in the short units that we examined is not interrupted by an insertion segment. Otherwise, the R loop maps of the long and short units do not differ significantly. The two classes therefore correspond to repeating units that do (IN+) or do not (IN-) contain an insertion segment. Models for the transcription and function of these two classes of repeating units are discussed.

Animals↗
PubMed1979

Genetics analysis of spore germination mutants of Bacillus subtilis 168: the correlation of phenotype with map location.

The isolation and characterization of 29 new germination (Ger) mutants of Bacillus subtilis 168 is described. These were classified, along with previously described mutants, into seven groups according to map location. The mutations in 26 GerA mutants mapped between cysB and thr; detailed mapping of two of these has located them very close to citG. These mutants were deficient in germination in alanine, but responded to the germinative combination of asparagine, glucose, fructose and KCl. One GerB mutant mapped on the origin-proximal side of hisA; it was normal in germination in alanine, but deficient in termination in a mixture of asparagine, glucose, fructose and KCl. Two GerC mutants were linked to lys, but were separable from a temperature-sensitive growth deficiency mapping between lys and trp. The GerC mutants had a similar germination phenotype to the GerA mutants. Three GerD mutants did not germinate in either of the above germinants or in Penassay Broth. They were located on the side of ery distal to cysA. The GerE mutant, which did not germinate in any of the three germinants, was located very close to citF and possessed an altered spore coat. The two GerF mutants were defective in germination in all three germinants and mapped on the origin proximal-side of hisA, but much closer to his than did the GerB mutant. A phosphoglycerate kinase-negative mutant altered in germination mapped between cysB and hisA (GerG). These mutants have established a minimum of seven locations important to germination, and will be useful in the development and appraisal of theories of spore germination.

Bacillus subtilis↗
PubMed1979

Fine structure of the 21S ribosomal RNA region on yeast mitochondria DNA. I. Construction of the physical map and localization of the cistron for the 21S mitochondrial ribosomal RNA.

1. We have used restriction enzyme analysis of petite mtDNAs to construct a detailed physical map of the 21S region on the mtDNA of the Saccharomyces cerevisiae strain JS1-3D. The map covers a segment of about 20,000 bp, on which the recognition sites of the enzymes HapII, HindII, HindIII, Sa1I, XhoI and HhaI have been localized (22 sites in total). This map has been checked in various ways against the independently constructed overall physical map of the mtDNA of strain JS1-3D. In addition, we have constructed a physical map with a resolution of about 200 bp of a HapII fragment of 1850 bp long, which carries the loci omega, RIB-1 and probably RIB-2. 2. The 21S rRNA hybridizes with the five adjacent HindII + III fragments TD9, DT19, TD15, DT14 and TT1, which lie in that order on the physical map of the 21S region. Of these, the two non-adjacent fragments TD9 and DT14 show a much stronger hybridization with 21S rRNA than DT19, TD15, and TT1. 3. The fragment DD5 (= DT19 + TD15) and part of DT14 belong to a sequence of about 1000 bp, which is absent from Saccharomyces carlsbergensis mtDNA. Although DD5 and DT14 show (very weak, respectively stronger) hybridization with 21S rRNA, the 1000 bp insert probably does not code for the 21S rRNA: the 21S rRNA of S. carlsbergensis comigrates with the 21S rRNA of JS1-3D on polyacrylamide gels under denaturing conditions. 4. Fragment DT14 hybridizes with the HindII + III fragment TD9, which shows the strongest hybridization with 21S rRNA. The presence of these sequence homologies has hampered the precise mapping of the 21S rRNA cistron. Our results are compatible, however, with the hypothesis that the sequences, coding for 21S rRNA, are located on HindII + III fragments that are not adjacent on JS1-3D mtDNA, namely TD9, DT14 and TT1.

DNA Restriction Enzymes↗
PubMed1978

Physical mapping of genes on yeast mitochondrial DNA: localization of antibiotic resistance loci, and rRNA and tRNA genes.

We have physically mapped the loci conferring resistance to antibiotics that inhibit mitochondrial protein synthesis (erythromycin, chloramphenicol and paromomycin) or respiration (oligomycin I and II), as well as the 21s and 14s rRNA and tRNA genes on the restriction map of the mitochondrial genome of the yeast Saccharomyces cerevisiae. The mitochondrial genes were localized by hybridization of labeled RNA probes to restriction fragments of grande (strain MH41-7B) mitochondrial DNA (mtDNA) generated by endonucleases EcoRI, HpaI, BamHI, HindIII, SalI, PstI and HhaI. We have derived the HhaI restriction fragment map of MH41-7B mit DNA, to be added to our previously reported maps for the six other endonucleases. The antibiotic resistance loci (antR) were mapped by hybridization of 3H-cRNA transcribed from single marker petite mtDNA's of low kinetic complexity to grande restriction fragments. We have chosen the single Sal I site as the origin of the circular physical map and have positioned the antibiotic loci as follows: C (99.5-1.Ou)--P (27-36.Ou)--OII (58.3-62u--OI (80-84u)--E (94.4-98.4u). The 21s rRNA is localized at 94.4-99.2u, and the 14s rRNA is positioned between 36.2-39.8u. The two rRNA species are separated by 36% of the genome. Total mitochondrial tRNA labeled with 125I hybridized primarily to two regions of the genome, at 99.5-11.5u and 34-44u. A third region of hybridization was occasionally detected at 70--76u, which probably corresponds to seryl and glutamyl tRNA genes, previously located to this region by petite deletion mapping.

Chloramphenicol↗
PubMed1978

Anatomy of herpes simplex virus (HSV) DNA. X. Mapping of viral genes by analysis of polypeptides and functions specified by HSV-1 X HSV-2 recombinants.

In an earlier paper (Morse et al., J. Virol 24:231--248, 1977) we reported on the provenance of the DNA sequences in 26 herpes simplex virus type 1 (HSV-1) X HSV-2 recombinants as determined from analyses of their DNAs with at least five restriction endonucleases. This report deals with the polypeptides specified by the recombinants and by their HSV-1 and HSV-2 parents. We have identified (i) the corresponding HSV-1 and HSV-2 polypeptides with molecular weights ranging from 20,000 to more than 200,000, (ii) the polypeptides that undergo rapid post-translational processing, and (iii) polypeptides that vary intratypically in apparent molecular weight. By comparing the segregation patterns of the polypeptides with those of the DNA sequence of the recombinants, we have mapped the templates specifying 26 polypeptides and several viral functions on the physical map of HSV DNA. The data show the following: (i) alpha polypeptides map at the termini of the L and S components of the HSV DNA. Although alpha ICP 27 maps entirely within the reiterated region of the L component, the template for alpha ICP 4 may lie only in part within the reiterated sequences of the S component. Of note is the finding that cells infected with a recombinant that contains both HSV-1 and HSV-2 DNA sequences in the S component produced alpha ICP 4 of both HSV-1 and HSV-2. (ii) Templates specifying beta and gamma polypeptides map in the L component and appear to be randomly distributed. (iii) Thymidine kinase and resistance to phosphonoacetic acid mapped in the L component. In addition, we have taken advantage of the rapid inhibition of host protein synthesis characteristic of HSV-2 infections and syncytial plaque morphology to also map the template(s) responsible for these functions in the L component. The implications of the template arrangement in HSV DNA are discussed.

Base Sequence↗