The ovalbumin gene: Cloning of the natural gene

The structural ovalbumin DNA sequences are not contiguous and are separated by multiple "intervening regions" in native chicken DNA. EcoRI, a restriction endonuclease that does not cleave the structural ovalbumin DNA sequences, digests the natural ovalbumin gene into three distinct fragments of 2.4, 1.8, and 9.5 kilobase pairs in length by cleaving within these "intervening regions." The 2.4-kilobase pair fragment contains only about 450 nucleotide pairs of coding sequence, with the rest being intervening sequences. This DNA fragment was cloned in bacteria by using the certified EK2 vector lambdagtWES.lambdaB after enrichment from total EcoRI-digested chicken DNA by a combination of RPC-5 column chromatography and preparative agarose gel electrophoresis. Five out of approximately 20,000 recombinant phage plaques were capable of hybridizing with a (32)P-labeled Hha I fragment of a recombinant plasmid pOV230 containing the entire structural ovalbumin gene. DNA amplified in these recombinant phages, lambdagtWES.OV2.4, was shown to contain the same restriction endonuclease cleavage sites as in the 2.4-kilobase pair EcoRI fragment previously determined by restriction mapping of total genomic chicken DNA. The intervening sequences were allowed to hybridize with excess total chicken DNA and oviduct nuclear RNA after nick-translation. They were found to be unique chicken DNA sequences, and appeared to be transcribed in their entireties during gene expression. Like the structural gene sequences, the expression of the intervening sequences is also inducible by steroid hormones.     

Mapping of the vaccinia virus thymidine kinase gene by marker rescue and by cell-free translation of selected mRNA

A selective plaque assay that uses thymidine kinase (TK)-deficient human 143 cells was developed to titer mixtures of TK(+) and TK(-) vaccinia virus. With this assay it could be shown that methotrexate-resistant TK(+) virus was formed in cells coinfected with TK(-) virus and wild-type virus DNA. By substituting vaccinia DNA fragments cloned in plasmids for virion DNA, this marker rescue system provided the basis for mapping the TK gene. Of the 15 HindIII fragments, only J could rescue five independently derived TK(-) mutants. This 5000-base-pair (bp) fragment maps approximately 80,000 bp from the left-end of the 180,000-bp vaccinia genome. Marker rescue could be detected with 18 ng or less of plasmid and was proportionate to DNA concentration. The resistance to methotrexate of the TK(+) recombinants was shown to be due to TK synthesis. Evidence that the HindIII J fragment contains the structural TK gene and not a regulatory element was demonstrated by the synthesis of active TK in a cell-free system programmed with mRNA selected by hybridization to the plasmid. Previous studies [Belle-Isle, H., Venkatesan, S. & Moss, B. (1981) Virology 112, 306-317] indicated that mRNAs coding for three immediate early polypeptides with molecular weights of 41,000, 21,000, and 17,000 map within HindIII J. The mapping of the easily selectable vaccinia virus TK gene now opens the way to genetic manipulations that should increase our understanding of vaccinia virus gene expression and facilitate the use of vaccinia virus as an efficient cloning vector for foreign genes.     

Isolation and mapping of plasmids containing the Salmonella typhimurium origin of DNA replication.

A purified EcoRI restriction endonuclease fragment that determines resistance to kanamycin and is incapable of self-replication was used to select autonomously replicating fragments from an EcoRI digest of a Salmonella typhimurium F' plasmid containing the chromosomal region believed to include the S. typhimurium origin of DNA replication. Both the F factor and S. typhimurium chromosome replication origins were cloned by this procedure. The EcoRI fragmentment containing the S. typhimurium origin of replication is 19.4 kilobase pairs long and includes functional asp+ and uncB+ genes. Restriction endonuclease analysis of deletions obtained from the S. typhimurium origin plasmid indicated that the replication origin (ori region) is contained within a 3.3-kilobase pair region. Comparison with Escherichia coli origin plasmids shows colinearity of gene arrangement on the chromosomes in this region and suggests that some, but not all, regions of the nucleotide sequence in the origin region may be conserved (identical) in these two bacterial species.     

Cloning of the silk fibroin gene and its flanking sequences.

Thirteen Escherichia coli clones containing the whole or a part of the fibroin gene (16 kilobases long) have been isolated. The starting material was DNA extracted from the posterior silk glands of Bombyx mori. Most clones were obtained from sheared DNA fragments linked by poly(dA)-poly(dT) joints to the plasmid pMB9. One of them includes the 5' end of the fibroin gene with a flanking sequence of 12 kilobases, and another includes the 3' end of the gene with a flanking sequence of about 1 kilobase. One clone was obtained by ligation, to pMB9, of a fragment generated by endodeoxy-ribonuclease EcoRI. This clone has a 21-kilobase insertion that probably includes the entire fibroin gene with flanking sequences at both ends. The cleavage sites for endodeoxyribonucleases EcoRI, HindIII, and BamHI have been established for the cloned sequences.     

Cmu gene rearrangement of mouse immunoglobulin genes in normal B cells occurs on both the expressed and nonexpressed chromosomes.

We have examined the organization of heavy-chain immunoglobulin genes on both the expressed and nonexpressed chromosomes of normal B lymphocytes from allotype heterozygous (BALB/c X C57BL/J)F1 mice. The C mu genes of BALB/c mice are on 12.4-kilobase EcoRI and 13.1-kilobase Kpn I restriction fragments, whereas those of C57BL/J mice are on 13.6-kilobase EcoRI and 14.3-kilobase Kpn I restriction fragments, allowing the examination of rearrangements on each chromosome independently. B lymphocytes from spleen and Peyer's patches expressing both IgD and IgM of the BALB/c allotype were isolated with a fluorescence-activated cell sorter. EcoRI and Kpn I restriction digests were hybridized with a C mu gene-containing probe. The C mu gene is present on both chromosomes. DNA rearrangements occur on both the expressed and nonexpressed chromosome within the 3.6-kilobase Kpn I/EcoRI restriction fragment containing the joining (JH) gene locus. We conclude that allelic exclusion of heavy-chain immunoglobulin gene expression is not mediated by JH-region DNA rearrangement of the expressed chromosome only. In contrast, analysis of the C kappa gene region from the same sorted B-cell DNA reveals a substantial quantity of germ-line context DNA. We also demonstrate that the deletions observed on the Eco RI fragment containing the C mu gene in myeloma cells and in C mu gene-containing recombinant DNAs do not usually occur in normally differentiating B lymphocytes and are likely to be confined to myeloma tumor cells.     

Molecular cloning of equine herpesvirus type 1 DNA: analysis of standard and defective viral genomes and viral sequences in oncogenically transformed cells.

Genomic DNA sequences of equine herpesvirus type 1 (EHV-1) have been cloned as BamHI and EcoRI restriction fragments into the plasmid pBR322 and propagated in Escherichia coli. With the exception of two EcoRI restriction fragments that reside in the S region of the viral genome, all of the cloned fragments demonstrated the same electrophoretic mobilities, restriction cleavage sites, and blot-hybridization patterns as did the parent fragments produced by BamHI or EcoRI digestion of virion DNA. The EcoRI J fragment and the BamHI E fragment of the L-region terminus were cloned after the addition of appropriate linker oligonucleotides. Fragments originating from each of the two isomeric forms of EHV-1 DNA were contained in this library of clones. Supramolar DNA fragments present only in the DNA of defective interfering (DI) particles of EHV-1 were generated from Bgl II digestion of DNA preparations enriched for EHV-1 DI particles and were cloned as Bgl II and EcoRI fragments into the plasmid vector. The cloned viral sequences represented in this defective genome mapped to the S region of EHV-1 DNA. Blot-hybridization analysis of EHV-1 transformed and tumor cell DNAs with the cloned BamHI B fragment confirmed that subgenomic viral sequences are present and indicated that those sequences map to the viral genome between 0.32 and 0.43 map unit.     

Insertion mutant of bacteriophage f1 sensitive to EcoRI.

The nucleotide sequence A-A-T-T was inserted into the intergenic region of the f1 genome at a site cleaved by Hae III (cleavage sequence (G-G-C-C). The resultant viable phage mutant (R199) contains a single site sensitive to the restriction endonuclease EcoRI (cleavage sequence G-A-A-T-T-C). This phage is sensitive to EcoRI restriction and modification in vivo and in vitro. Its potential for use as a cloning vector has been tested by construction in vitro of an f1/pBR322 chimeric phage. The four bases inserted into wild-type f1 to generate the R199 mutant came from a small restriction fragment obtained by digesting plasmid pBR322 with EcoRI and HindIII. The use of this linker prepared from a biological substrate is an example of a technique for constructing restriction enzyme sites in vitro. It is presented as an alternative to the use of synthetic linkers and should be generally applicable.     

Cleavage of Replicating Forms of Mitochondrial DNA by EcoRI Endonuclease

Digestion of mouse L cell mitochondrial DNA with EcoRI restriction endonuclease produces two linear duplex fragments comprising 86.3 +/- 2.0% and 14.2 +/- 1.0% of the circular genome length (16,000 +/- 470 nucleotide pairs). Digestion of human HeLa cell mitochondrial DNA with EcoRI produces three linear duplex fragments comprising 49.2 +/- 1.0%, 44.4 +/- 0.9%, and 6.4 +/- 0.4% of the circular genome length (16,590 +/- 710 nucleotide pairs). These fragments are shown to be generated by cleavage in unique regions of the mouse and human mitochondrial DNAs. An electron microscopic analysis of partially replicated molecules cleaved by EcoRI establishes a unidirectional mode of DNA replication for L cell mitochondrial DNA. The origin for DNA replication is located on the larger EcoRI fragment at a position that is 1,890 +/- 250 nucleotide pairs (11.8 +/- 1.2% of the circular genome length) from the proximal restriction site. Replication proceeds unidirectionally away from this restriction site throughout the length of the larger EcoRI fragment. Analysis of L cell, D-loop mitochondrial DNA cleaved by EcoRI indicates that a unique sequence is synthesized in formation of the D-loop in these nonreplicating molecules. The origin of D-loop synthesis is located on the larger EcoRI fragment at a position 1,760 +/- 180 nucleotide pairs (11.0 +/- 1.1% of the circular genome length) from the proximal restriction site and is, therefore, the origin for unidirectional displacement replication.     

Cloning of developmentally regulated flagellin genes from Caulobacter crescentus via immunoprecipitation of polyribosomes.

Immunoprecipitation of Caulobacter crescentus polyribosomes with antiflagellin antibody provided RNA for the synthesis of cDNA probes that were used to identify three specific EcoRI restriction fragments (6.8, 10, and 22 kilobases) in genomic digests of Caulobacter DNA. The RNA was present only in polyribosomes isolated from a time interval in the Caulobacter cell cycle that was coincident with flagellin polypeptide synthesis. The structural gene for Mr 27,500 flagellin polypeptide was assigned to a region of the 10-kilobase EcoRI restriction fragment by DNA sequence analysis. Analysis of mutants defective in motility further established a correlation between the Mr 27,500 flagellin gene and the flaE gene locus [Johnson, R. C. & Ely, B. (1979) J. Bacteriol. 137, 627-634]. The other EcoRI fragments that hybridize with the immunoprecipitated polyribosome-derived cDNA probe are also temporally regulated and have features that suggest they encode other polypeptides associated with the flagellum. Modifications were required to adapt the procedure of immunoprecipitation of polyribosomes for use with Caulobacter and should be applicable to the production of specific structural gene probes from other prokaryotic systems.     

Molecular cloning and sequence determination of the nuclear gene coding for mitochondrial elongation factor Tu of Saccharomyces cerevisiae.

A 3.1-kilobase Bgl II fragment of Saccharomyces cerevisiae carrying the nuclear gene encoding the mitochondrial polypeptide chain elongation factor (EF) Tu has been cloned on pBR327 to yield a chimeric plasmid pYYB. The identification of the gene designated as tufM was based on the cross-hybridization with the Escherichia coli tufB gene, under low stringency conditions. The complete nucleotide sequence of the yeast tufM gene was established together with its 5'- and 3'-flanking regions. The sequence contained 1,311 nucleotides coding for a protein of 437 amino acids with a calculated Mr of 47,980. The nucleotide sequence and the deduced amino acid sequence of tufM were 60% and 66% homologous, respectively, to the corresponding sequences of E. coli tufA, when aligned to obtain the maximal homology. Plasmid YRpYB was then constructed by cloning the 2.5-kilobase EcoRI fragment of pYYB carrying tufM into a yeast cloning vector YRp-7. A mRNA hybridizable with tufM was isolated from the total mRNA of S. cerevisiae D13-1A transformed with YRpYB and translated in the reticulocyte lysate. The mRNA could direct the synthesis of a protein with Mr 48,000, which was immunoprecipitated with an anti-E. coli EF-Tu antibody but not with an antibody against yeast cytoplasmic EF-1 alpha. The results indicate that the tufM gene is a nuclear gene coding for the yeast mitochondrial EF-Tu.     

Intracisternal A-particle genes: identification in the genome of Mus musculus and comparison of multiple isolates from a mouse gene library.

The genome of Mus musculus contains multiple copies (500 -1000) of DNA sequences related to the 35S RNA of intracisternal type A particles (IAPs). Using labeled IAP RNA as a probe in blot-hybridization experiments, we have identified a characteristic electrophoretic pattern of reactive fragments generated by restriction endonuclease cleavage of mouse DNA. From the genomic blots, we deduced a composite restriction map for a 6.5- to 7-kilobase (kb) DNA region containing sequences homologous to the IAP RNA. Units of this type appeared to be interspersed without obvious regularity in nonhomologous flanking regions. A 5.2-kb segment of this unit was inserted directly into plasmid pBR322 from HindIII/EcoRI digest of mouse DNA. The fragment was cloned and then labeled by nick-translation and used to scan a mouse embryo gene library (average 16-kb inserts in lambda Charon 4A); 1% of the library samples hybridized, confirming the extensive reiteration of IAP genetic units. Among six different library isolates containing 6.5- to 7-kb IAP units, some restriction sites were highly conserved whereas others varied in both occurrence and position. Despite this variation, heteroduplexes between the individual isolates showed continuous IAP homology regions of 7 kb. No flanking region homologies were seen in this limited sample. Some evidence suggests that mouse DNA may contain other dispersed sequence elements related to but smaller than the genetic unit defined above.     

Molecular cloning of the mouse ouabain-resistance gene.

DNA prepared from ouabain-resistant mouse cells was able to transform ouabain-sensitive CV-1 cells to ouabain resistance after DNA-mediated gene transfer. The murine DNA fragment responsible for ouabain resistance was detected on the background of CV-1 DNA by virtue of a repetitive DNA sequence element that reacts positively with a mouse repeat DNA clone. CV-1 DNA is nonreactive with this probe. Southern analysis of several independently derived ouabain-resistant transformants indicates that the mouse ouaR gene is located on a 6.5-kilobase EcoRI restriction fragment. The 6.5-kilobase DNA fragment was initially isolated from a lambda phage library made from a ouabain-resistant secondary transformant and subsequently was subcloned in the plasmid vector pAT153. This plasmid was able to transform wild-type CV-1 cells to ouabain resistance at a frequency of about 10 cells per ng of DNA.     

Cloning and mapping of the replication origin of Escherichia coli.

The replication origin of Escherichia coli has been cloned on a nonreplicating DNA fragment coding for ampicillin resistance. This recombinant DNA, named pSY211, replicates depending on the presence of the replication origin and can be recovered as a closed circular plasmid DNA of 10.7 megadaltons (Mdal). A restriction map has been constructed. EcoRI cleaves pSY211 into two fragments: one is the ampicillin fragment of 4.5 Mdal and the other is a chromosomal fragment of 6 Mdal and contains the origin. The 6 Mdal EcoRI fragment has four BamHI sites, three HindIII sites, and one Xho I site. A mutant of pSY211 has been isolated which is lacking two BamHI fragments of the chromosomal fragment. In recA hosts, pSY211 is lost at a high frequency. In recA+ hosts, pSY211 is integrated into the chromosome due to nucleotide sequence homology between pSY211 and the replication origin of the E. coli chromosome. The integration site has been mapped. We conclude that the replication origin is located at a site between uncA and rbsK, at about 83 min on the genetic map of E. coli.     

Isolation and characterization of the mouse metallothionein-I gene.

Double-stranded cDNA was synthesized from a mouse liver mRNA fraction enriched for metallothionein mRNA activity, ligated to restriction site linkers, inserted into pBR322, and used to transform Escherichia coli chi 1776. The sequence of the largest plasmid containing DNA that hybridized to metallothionein mRNA was determined and shown to contain a 380-base-pair insert that includes the entire coding region and 3' untranslated region of metallothionein-I. The metallothionein-I insert was nick-translated and used to screen both a mouse myeloma and a mouse embryo DNA library in bacteriophage lambda. A metallothionein-I genomic clone containing 13-15 kilobase pairs of mouse DNA was isolated from each library. Both contain a 3.8-kilobase-pair EcoRI fragment that hybridizes to the metallothionein-I probe. The location, size, and orientation of the metallothionein-I gene within the 3.8-kilobase-pair fragment were determined by heteroduplex and restriction mapping. The gene spans 1.1 kilobase pairs and contains at least two introns.     

Bovine galactosyltransferase: identification of a clone by direct immunological screening of a cDNA expression library.

A 1.3-kilobase cDNA clone (7A) coding for bovine galactosyltransferase (glycoprotein 4-beta-galactosyltransferase, EC 2.4.1.38) was isolated from a lambda gt11 expression library by immunological screening with monospecific polyclonal antisera to the affinity-purified bovine enzyme. The nucleotide sequence of this clone predicts an open reading frame that starts at the 5' end of the insert and codes for a polypeptide of 334 amino acids with Mr 37,645. Based on a Mr of 57,000 for the membrane-bound enzyme this clone accounts for approximately 61% of the coding sequence. Portions of the predicted amino acid sequence matched the six tryptic peptides isolated from affinity-purified bovine galactosyltransferase. Clone 7A hybridizes to a 4.8-kilobase bovine mRNA and identifies multiple EcoRI restriction fragments in bovine, murine, and human DNA.     

Molecular cloning and cell cycle-specific regulation of a functional human thymidine kinase gene.

A functional thymidine kinase (TK; ATP:thymidine 5'-phosphotransferase, EC 2.7.1.21) gene has been molecularly cloned from human DNA. The gene was rescued from a genomic library of TK-deficient mouse L cells transformed to the TK+ phenotype with total HeLa cell DNA. Of 14 overlapping clones, only one contained the intact human TK gene. The cloned recombinant bacteriophage carries a 16-kilobase insert derived entirely from human DNA and is capable of transforming LTK- cells to TK+ with an efficiency of 10 TK+ colonies per ng of DNA per 10(6) cells. Restriction endonuclease mapping shows that the functional human TK gene is at least twice as long as that reported for chicken. A 1.6-kilobase Xho I/EcoRI fragment was subcloned and found to hybridize to a human mRNA of 1.5 kilobases. When introduced into LTK- cells, the cloned human TK gene is regulated in the cell cycle-specific manner characteristic of TK+ mammalian cells. That is, TK activity in synchronized cells increases markedly with the onset of DNA synthesis. The signals governing the S-phase induction of TK activity reside within 16 kilobases of human DNA and are correctly interpreted by mouse cells.     

Human papillomavirus DNA: physical map.

Human papillomavirus (HPV) DNA form I (supercoiled) was prepared from plantar warts. HPV DNA was cleaved with restriction enzymes obtained from the following sources: escherichia coli (EcoRI), Hemophilus influenzae strain Rd (both unfractionated Hind and aeparated HindII and HindIII enzymes) and Hemophilus parainfluenzae (HpaI). The cleavage products were analyzed by polyacrylamide gradient slab gel electrophoresis and electron microscopy. HPV DNA was cleaved into two fragments by EcoRI (87% and 13% of the genome) and into six fragments, ranging in size from 33.5 to 1.2% of the genome, by Hind endonucleases. The six Hind fragments result from the cleavage of three sequences recognized by HindII, two of which are also cleaved by HpaI, and of three sequence recognized by HindIII. The order of these fragments was determined by comparing their size with that of the fragments obtained with HindII, HindIII, HpaI, and the mixture of HindIII + Hpal. The two EcoRI cleavage sites were located on two adjacent Hind fragments and one of these sites has been taken for the zero point to construct a physical map. The treatment of superhelical HPV DNA with bacteriophage T4 gene 32 protein yields circular structures with a denaturation loop. The cleavage of these complexes with EcoRI and HindIII has shown two easily denatured regions which were located on the cleavage map.     

Cloning and localization of the lepidopteran protoxin gene of Bacillus thuringiensis subsp. kurstaki.

Bacillus thuringiensis subsp. kurstaki produces a proteinaceous crystalline inclusion that is toxic for lepidopteran larvae. There are several size classes of plasmids in this organism and the presence of one or more has been correlated with production of this protein, defined as a protoxin. DNA fragments of B. thuringiensis subsp. kurstaki, obtained by EcoRI digestion, were cloned into the vector Charon 4A. Recombinant phage were screened immunologically for the production of protoxin. Cells infected with one phage, C4K6c, produced antigen that was the same size as the protoxin and was toxic to Manduca sexta larvae. A 4.6-kilobase-pair (kbp) EcoRI fragment from C4K6c was subcloned into pBR328 and in both orientations in pHV33. Both Escherichia coli and Bacillus subtilis containing these recombinant plasmids produced antigen that crossreacted with antibody directed against the protoxin. The various sized plasmids of B. thuringiensis were purified and only an EcoRI fragment from the 45-kbp plasmid hybridized to phage C4K6c. One of the pHV33 subclones, pSM36, hybridized to the same size EcoRI/HindIII restriction fragments from plasmid or chromosomal DNA. The cloned EcoRI fragment contained a 0.9-kbp Pvu II fragment that was also present in chromosomal but not in plasmid digests. The original clone was therefore of chromosomal origin, although very similar or identical protoxin genes were present in both the 45-kbp plasmid and the chromosome. Several acrystalliferous nontoxic mutants have been isolated that lacked the 45-kbp plasmid and in some cases all plasmids. All of the mutants contained the chromosomal gene but did not produce protoxin antigen.