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KpnI and SstI fragments representing 96% of the varicella-zoster virus genome, including the termini, were cloned in plasmid vector pAT153. The clones were used to derive maps of virion DNA for SstI, KpnI, XhoI, PvuII, EcoRI and SalI by molecular hybridization and restriction endonuclease digestion.
We have studied the organization of the human immunoglobulin heavy-chain genes by pulse field gel electrophoresis as well as by isolation of cosmid clones. The total length of the heavy-chain variable region locus was estimated to be approximately 3000 kb. We found that D segments including a recently isolated D5 segment were dispersed among VH segments. We identified a pseudo V segment 18 kb 3' to the D5 segment in isolated cosmid clones. A 300 kb fragment produced by MluI digestion contained VH, D, JH segments and the distance between VH and D was estimated to be approximately 240 kb. Overlapping cosmid clones containing the human D1, D2, D3, D4, JH, Cmu and C delta genes were isolated. Restriction maps of these regions indicated that the distance between D and JH is about 22 kb. A partial restriction map of the VH locus was constructed using the pulse field gel electrophoresis technique and deletion of VH segments in B cells.
A physical map of the composite R plasmid NR1 has been constructed using specific cleavage of deoxyribonucleic acid (DNA) by the restriction endonuclease EcoR-. Digestion of composite NR1 DNA by EcoRI yields thirteen fragments. The six largest fragments (designated A to F) are from the resistance transfer factor component that harbors the tetracycline resistance genes (RTF-TC). The seven smallest fragments (designated G to M) are from the r-determinants component that harbors the chloramphenicol (CM), streptomycin-spectinomycin (SM/SP), and sulfonamide (SA) resistance genes. The largest fragment of several RTF-TC segregants of NR1 that have deleted the r-determinants component is 0.8 X 10(6) daltons larger than fragment A of composite NR1. Only a part of fragment H of the r-determinants component is amplified in transitioned NR1 DNA in Proteus mirabilis, which consists of multiple, tandem sequences of r-determinants attached to a single copy of the RTF-TC component. Both of these changes can be explained by the locations of the excision sites at the RTF-TC: r-determinants junctions that are involved in the dissociation and reassociation of the RTF-TC and r-determinants components. The thirteen fragments of composite NR1 DNA produced by EcoRI have been ordered using partial digestion techniques. The order of the fragments is: A-D-C-E-F-B-H-I-L-K-G-M-J. The approximate locations of the TC, CM, SM/SP, and SA resistance genes on the EcoRI map were determined by analyzing several deletion mutants of NR1.
A physical and genetical map of the plasmid pEA3 indigenous to Enterobacter agglomerans is presented. pEA3 is a 111-kb large plasmid containing a 23-kb large cluster of nif genes which shows extensive homology (Southern hybridization and heteroduplex analysis) to the entire nif gene cluster of Klebsiella pneumoniae (Kp) M5a1. All the nif genes on pEA3 are organized in the same manner as in K. pneumoniae, except nifJ, which is located on the left end of pEA3 nif gene cluster (near nifQB). A BamHI restriction map of pEA3 and a detailed restriction map of the 23-kb nif region on pEA3 is also presented. The nif genes of pEA3 showed a low level of acetylene reduction in Escherichia coli, demonstrating that these genes are functional and contain the whole genetic information required to fix nitrogen. The origin of vegetative replication (OriV) of pEA3 was localized about 5.5 kb from the right end of the nif gene cluster. In addition to pEA3, large plasmids from four other strains of E. agglomerans showed homology to all the Kp nif genes tested, indicating that in diazotrophic strains of E. agglomerans nif genes are usually located on plasmids. In contrast, in most of the free-living, nitrogen-fixing bacteria the nif genes are on chromosome.
A whole-genome restriction map of Deinococcus radiodurans, a radiation-resistant bacterium able to survive up to 15,000 grays of ionizing radiation, was constructed without using DNA libraries, the polymerase chain reaction, or electrophoresis. Very large, randomly sheared, genomic DNA fragments were used to construct maps from individual DNA molecules that were assembled into two circular overlapping maps (2.6 and 0.415 megabases), without gaps. A third smaller chromosome (176 kilobases) was identified and characterized. Aberrant nonlinear DNA structures that may define chromosome structure and organization, as well as intermediates in DNA repair, were directly visualized by optical mapping techniques after gamma irradiation.
We constructed a yeast artificial chromosome (YAC) contig spanning the genes encoding Kit (Kit), the platelet-derived growth factor alpha receptor (Pdgfra), and fetal liver kinase 1 (Flk1), three members of a receptor tyrosine kinase gene family located in the central portion of mouse chromosome 5. The orientation of YAC clones and the extent of their overlap was determined by "probe content mapping," that is, hybridization analysis of YAC clones using the available gene probes and YAC end sequences. For four YAC clones, which constitute a minimal set spanning 1.8 Mb, a detailed restriction map was constructed. This map, in conjunction with the previously published long-range restriction map, indicates the order, the physical distances, and the relative transcriptional orientations of the Pdgfra, Kit, and Flk1 genes. The YAC clones and corresponding YAC end probes presented here provide an important resource for the molecular analysis of a cluster of developmental mutations, namely dominant white spotting (W), patch (Ph), recessive spotting (rs), and rump-white (Rw), associated with this chromosomal region.
A mutant EcoRI endonuclease restriction site has been identified in 3 of 37 Black subjects and in 2 sibs of one of these persons. This mutation was not encountered in 13 Whites. It is located approximately 6 kilobases "inside" the normal site in the 5' flanking sequence of the alpha-globin chain complex. The shortened alpha-globin gene-bearing segment produced in the EcoRI digest produces a restriction map similar to that observed for the common alpha-globin gene deletion observed in the Black population. However, the restriction map with BamHI is normal, confirming that all four alpha loci are present.
The positive regulatory gene (phoM) for alkaline phosphatase of Escherichia coli was cloned on a mini-F plasmid pMF3 from the E. coli chromosome by a shotgun method. The hybrid plasmid pTHR32, which carries 10.8 kb chromosomal DNA, complemented both phoM and thrB mutations. The restriction map was constructed. Based upon this information, several PhoM- deletion plasmids and smaller PhoM+ plasmids were constructed in vitro. By examining the phenotypes and the physical maps of these plasmids, we could define the phoM gene locus in a 2.5 kb region on the restriction map of the cloned chromosomal DNA fragment. The PhoM+ plasmid not only enabled a phoM- -phoR- double mutant to express phoA (the structural gene for the alkaline phosphatase) but also phoB (another positive regulatory gene for phoA). These results are consistent with a model for genetic regulation of phoA expression that proposes that both the phoM and phoR gene products activate phoB expression under phosphate starved conditions, and PhoB protein, in turn, activates phoA expression. The phoM gene product was identified by the maxicell method as a protein with a molecular weight of 60,000. A hybrid plasmid that carries a phoM'-'lacZ fused gene on mini-F vector pMF3 was constructed in vitro. This plasmid enabled us to study phoM gene expression by measuring the beta-galactosidase level in the cells. The plasmid was introduced into various regulatory mutants related to the phosphate regulon, and phoM gene expression in these strains was studied under conditions of limited or excess phosphate. It was found that phoM expression was not regulated by phosphate nor by any of the pho genes. The transcriptional direction of phoM was found to be clockwise toward the thr operon on the E. coli genetic map. The fusion gene product interfered with phoB and phoA expression in the phoR mutants. Overproduction of PhoM protein increased phoB and phoA expression only in the phoR mutants. The implications of these findings are discussed.
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Mutants of Anabaena sp. strain PCC 7120 unable to grow aerobically on dinitrogen were isolated by mutagenesis with UV irradiation, followed by a period of incubation in yellow light and then by penicillin enrichment. A cosmid vector, pRL25C, containing replicons functional in Escherichia coli and in Anabaena species was constructed. DNA from wild-type Anabaena sp. strain PCC 7120 was partially digested with Sau3AI, and size-fractionated fragments about 40 kilobases (kb) in length were ligated into the phosphatase-treated unique BamHI site of pRL25C. A library of 1,054 cosmid clones was generated in E. coli DH1 bearing helper plasmid pDS4101. A derivative of conjugative plasmid RP-4 was transferred to this library by conjugation, and the library was replicated to lawns of mutant Anabaena strains with defects in the polysaccharide layer of the envelopes of the heterocysts. Mutant EF116 was complemented by five cosmids, three of which were subjected to detailed restriction mapping; a 2.8-kb fragment of DNA derived from one of the cosmids was found to complement EF116. Mutant EF113 was complemented by a single cosmid, which was also restriction mapped, and was shown to be complemented by a 4.8-kb fragment of DNA derived from this cosmid.
A rapid, convenient and economical method for the hybridization of electrophoretically resolved RNA to DNA restriction fragments immobilized on nitrocellulose filters is described. DNA was digested, electrophoresed on agarose gels in a wide band and transferred to a nitrocellulose filter. The filter was then placed on the surface of a second gel containing radioactively labeled RNA electrophoresed under denaturing conditions in a similar way. The filter and gel were oriented so that the DNA and RNA bands were perpendicular to one another and the RNA was transferred from the gel through the filter under conditions which promote RNA-DNA hybridization. Following washing, the filter was autoradiographed. RNA-DNA sequence relationships could be conveniently determined from the spots produced at regions of intersection of homologous nucleic acids. The two dimensional array formed in this procedure fascilitates the rapid ordering of DNA restriction fragments. An example of its use for this purpose is presented.
An efficient procedure combining the Southern Cross method described by H. Potter and D. Dressler (1986, Gene 48, 229-239) and the use of nonradioactive reporter molecules which allows fast restriction mapping of DNA molecules up to 40 kb is described. In this method, photobiotin-labeled fragments for one restriction enzyme cross-hybridize with at least five unlabeled digests transferred onto nylon membranes. Hybridization spots are revealed on recipient membranes at a homologous fragment cross, allowing direct restriction map construction. In comparison with 32P "cross-blot" hybridization, photobiotin labeling is safer, faster, and easier to dispose of.
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We present the first complete map of the Haemophilus influenzae genome, consisting of a detailed restriction map with a number of genetic loci. All of the ApaI, SmaI, and RsrII restriction sites (total of 45 sites) were mapped by Southern blot hybridization analysis of fragments separated by pulsed-field gel electrophoresis. Cloned genes were placed on the restriction map by Southern hybridization, and antibiotic resistance loci were also located by transformation with purified restriction fragments. The attachment site of the HP1 prophage was mapped. In addition, the number, locations, and orientations of the six rRNA operons in the H. influenzae chromosome were determined. The positions of conserved restriction sites in these rrn operons confirm that the direction of transcription is 16S to 23S, as in most other bacteria. The widely used strain BC200 appears to contain an unexpected 45-kilobase duplication.
Unintegrated linear and closed circular DNAs of B- and N-tropic endogenous BALB/c murine leukemia virus (MuLV) were extracted from newly infected mouse cells and cleaved with EcoRI, XhoI, PvuI, HindIII, SalI, XbaI, KpnI, SmaI, and PstI restriction endonucleases. The DNA fragments were separated by electrophoresis and analyzed by the Southern blot hybridization procedure. EcoRI did not cleave the two genomes. A physical map of 15 cleavage sites on B- and N-tropic genomes was constructed with the other restriction endonucleases. Identical cleavage sites of B- and N-tropic MuLV DNAs were found with all these enzymes. However, the N-tropic linear genome was found to lack about 75 base pairs at each end of the molecule. PstI, KpnI, and SmaI recognize a cleavage site at both ends of the linear molecules. And sequences derived from the 5' end of the RNA genome were found in the third left end of the linear DNA and at its extreme right-end terminus, suggesting the presence of redundant sequences. Two species of closed circular viral DNA were observed. The larger species has the same size as the linear molecule and appears to be a circularized form of linear DNA. The smaller species contains sequences common to both the linear and the larger circular viral DNA but seems to be deleted from sequences present at either one or both ends of the linear DNA. Therefore, the general structure of the linear and circular DNA species of these B- and N-tropic endogenous BALB/c MuLV appears analogous to the structure found with other retroviruses.
We present the locations of the cleavage sites for the BamI, KpnI, and SalI restriction endonucleases within the DNA molecules of herpes simplex virus type 1 (HSV-1) strains Justin and F. These restriction enzymes cleave the HSV-1 DNA at many sites, producing relatively small fragments which should prove useful in future studies of HSV-1 gene structure and function. The mapping data revealed the occurrence of heterogeneity within three regions of the viral genome including (i) the region spanning map coordinates 0.74--0.76, (ii) the ends of the large (L) DNA component, and (iii) the junction between the large (L) and the small (S) components. The heterogeneity in the ends of L and the S-L junctions of HSV-1 (Justin) and HSV-1 (F) DNAs was grossly similar to that previously reported to occur in the ends of L and the S-L junctions of the HSV-1 (KOS) DNA (M. J. Wagner and W. C. Summers, J. Virol. 27:374--387, 1978). Thus, cleavage of these regions with restriction endonucleases yielded sets of minor fragments differing in size by constant increments. However, the various strains of HSV-1 differed with respect to the numbers, size increments, and relative molarities of the various minor fragments, suggesting that the parameters of the heterogeneity are inherited in the structural makeup of the HSV-1 genome. The strain dependence of the pattern of heterogeneity can be most easily explained in terms of variable sizes of the terminally reiterated a sequence, contained in the DNA molecules of these three strains of HSV-1.
Fourteen different plasmids hybridizing to Aspergillus nidulans 5S rRNA were isolated from a gene bank obtained after cloning Sau3A partial digests of A. nidulans DNA in a yeast--Escherichia coli vector, pBB29. The restriction maps of these plasmids were determined. The size of the cloned fragments was 2.7-9.5 kb, 12 of the plasmids were found to code for single 5S rRNA genes and 2 coded for 2 genes. No similarity of the sequences surrounding the 5S rRNA genes was found by restriction mapping.
One of the most important prognostic factors in neuroblastoma is amplification of the MYCN gene, which is strongly associated with advanced stages of disease and a poor prognosis. Although the MYCN amplicon sometimes spans more than 1 Mb, no other consistently expressed sequences from the MYCN amplicon have been reported. However, DDX1, a gene encoding a DEAD box protein, was recently mapped to chromosome 2p24 and is frequently co-amplified with MYCN. Therefore, we performed genomic mapping with YACs to determine the physical relationship between DDX1 and MYCN, and whether DDX1 was contained within the core region of amplification. Based on YAC restriction mapping and content analysis, DDX1 maps 340 kb 5' of MYCN, outside the core domain of consistent amplification. Interestingly, we also determined by sequence analysis and detailed restriction mapping that G21, previously isolated as a 'neuroblastoma-specific' cDNA clone from an MYCN amplicon, is a partial cDNA of DDX1. Our data confirm that DDX1 is amplified in some but not all MYCN-amplified tumors, and that it is rearranged in other cases. This suggests that the co-amplification of DDX1 is due to its proximity to MYCN.