An amplified insect dihydrofolate reductase gene contains a single intron

We have used methotrexate-resistant mosquito (Aedes albopictus) cells as the source of DNA for cloning an 8.5-kb EcoRI fragment containing an amplified dihydrofolate reductase (DHRF) gene. An estimated 1200 copies of the DHFR gene were represented in nuclear DNA from Mtx-5011-256 cells, which were 3000-fold more resistant to methotrexate than wild-type cells. Southern blot analysis indicated that all of the amplified DHFR genes were contained within a 1.8-kb AccI fragment represented in the cloned DNA. In contrast to mammalian DHFR genes which span approximately 30 kb, the complete amino acid coding sequence of the mosquito DHFR gene spanned 614 nucleotides, including a single 56-nucleotide intron that interrupted a conserved Arg codon at amino acid position 27. Additional introns characteristic of mammalian DHFR genes were absent; conservation of the first intron in the mosquito DHFR gene supports a regulatory role for this intron. The mosquito DHFR gene coded for a 186-amino-acid protein with 43-48% similarity to vertebrate DHFR.     

Genetic changes in methotrexate-resistant mosquito cells.

A stepwise selection procedure was used to obtain from Mtx-5011 Aedes albopictus cells, variants with increased resistance to methotrexate (mtx). On the basis of growth, the Mtx-5011 derivatives were 270- to 3,000-fold more resistant to mtx than wild-type mosquito cells. Properties associated with mtx resistance in these cells were consistent with amplification of the dihydrofolate reductase (DHFR) gene. The cells overproduced DHFR protein, were enriched with DHFR mRNA, and DNA from resistant cells was enriched for a band that likely contained the DHFR coding sequence. Karyotype analysis indicated that high levels of resistance were accompanied by a conversion to tetraploidy, chromosome rearrangements, and an apparent duplication of one of the mosquito chromosomes.     

DNA fragments from regions involved in surface antigen expression specifically identify Listeria monocytogenes serovar 4 and a subset thereof: cluster IIB (serotypes 4b, 4d, and 4e).

Listeria monocytogenes serotype 4b has frequently been implicated in sporadic as well as epidemic listeriosis. On the basis of pulsed-field fingerprinting, serotype 4b strains, along with strains of serotypes 4d and 4e, constitute one genomic cluster (IIB). We have identified two genomic regions essential for the expression of surface antigens which previously were shown to be specific to cluster IIB strains. A DNA probe of 1.1 kb derived from one of the regions (probe 1) hybridized only with strains of serotypes 4b, 4d, and 4e in Southern blots and dot blots. A different DNA probe of 0.3 kb (probe 2), derived from the other region, hybridized with all serovar 4 strains (serotypes 4b, 4a, 4c, 4d, and 4e). All other L. monocytogenes serotypes were negative with probe 1 or 2. Use of probe 1 in Southern blots of EcoRI-digested genomic DNA revealed a restriction fragment length polymorphism in serotype 4b strains, with the hybridizing EcoRI fragments being 4.5 kb (strains of the epidemic clone) and either 4.5 or 5.0 kb (all other serotype 4b strains). Although the probes hybridized with a special group of Listeria innocua strains which also expressed the surface antigens, the latter could be readily distinguished by the size of the hybridizing EcoRI fragment with probe 1 (ca. 2.2 kb). These data suggest that the combined use of these probes with L. monocytogenes can readily and specifically identify cluster IIB strains as well as the entire serovar 4 complex.     

Organization of delta-crystallin genes in the chicken.

Double-stranded DNA was synthesized from delta-crystallin mRNA prepared from lens fibers of 15-day-old chick embryos and cloned at the Pst I site of the plasmid pBR322. Using the cloned cDNA and single-stranded cDNA as hybridization probes, a number of genomic DNA fragments containing delta-crystallin gene sequences have been cloned from the partial and complete EcoRI digests of chick brain DNA. One of the clones from the partial digests contains a DNA fragment that consists of four EcoRI fragments of 7.6 kb, 4.0 kb, 2.6 kb, and 0.8 kb. The gene sequences reside in the (5')7.6 kb - 0.8 kb - 4.0 kb (3') fragments. Electron microscopy has provided evidence that the cloned DNA fragment includes the entire gene sequences complementary to delta-crystallin mRNA except for the 3' terminal poly(A) tail, and that the delta-crystallin gene is interrupted by at least 13 intervening sequences. Another clone contains a genomic fragment that consists of two EcoRI fragments of 3.0 kb and 11 kb. The DNA fragment in the latter clone represents a different delta-crystallin gene, as judged by restriction endonuclease mapping and by electron microscopy.     

Molecular cloning of genomic DNA segments partially coding for human thymidylate synthase from the mouse cell transformant

Mouse cells deficient in the enzyme thymidylate synthase [TS; EC 2.1.1.45] were serially transformed with human DNA to yield primary and secondary transformants which produced human TS [Ayusawa, D., Shimizu, K., Koyama, H., Takeishi, K., & Seno, T. (1983) J. Biol. Chem. 258, 48-53]. Southern blot hybridization of their genomic DNA showed that six secondary transformants examined contained in common a 5.5 kb EcoRI fragment hybridized with a human Alu sequence. From the secondary transformant genomic library constructed with phage lambda Charon 4A, two recombinant phage clones carrying Alu sequences were isolated. Restriction endonuclease mapping revealed that the insert DNAs of the two phage clones overlapped and covered a region of 19 kb in total. Within this region at least six Alu sequences were located. A 2.0 kb DNA fragment, prepared from an EcoRI fragment subcloned in plasmid pBR322 and free of Alu sequences, hybridized to a single band on RNA blots of primary and secondary transformant poly(A)+ RNA, but not to RNA of mouse wild-type and recipient cell lines. The relative amount of the presumed human TS mRNA was linearly correlated with the relative activity of human TS in various types of mouse transformant cells. These results indicate that these two phage clones contain genomic DNA sequences encoding human TS.     

Organization of a cloned repetitive DNA fragment in mosquito genomes (Diptera: Culicidae).

The structure and genomic organization of a cloned 5.2-kb repetitive DNA fragment, H-85, isolated from the Aedes albopictus genome have been examined. In situ hybridization of the 3H-labeled H-85 DNA to the meiotic and mitotic chromosome preparations of Ae. albopictus shows that the sequences homologous to H-85 DNA are dispersed throughout the length of all three pairs of chromosomes. A similar pattern of in situ hybridization appears in Aedes seatoi, Aedes flavopictus, and Aedes aegypti. The study shows that the arrangement of sequences in the cloned 5.2-kb fragment is rare in the Ae. albopictus genome. Dot-blot hybridization reveals that the sequences homologous to H-85 DNA are present in 12 species of mosquitoes examined, belonging to six genera in subfamilies Culicinae ad Anophelinae. The H-85 sequences are also present in the genome of Mochlonyx velutinus of the nematocerous family Chaoboridae, earlier proposed as the ancestor of the mosquito family Culicidae. Although the sequences homologous to H-85 DNA are present in different species of mosquitoes, they have diverged in their structure and organization. The cloned 5.2-kb fragment is composed of elements of different and independently evolving repetitive DNA families.     

Multiple copies of IS256 in staphylococci.

EcoRI-digested cellular DNAs of 150 staphylococcal clinical isolates were probed with a plasmid containing a DNA piece from within the open reading frame of IS256. Most of the Gmr staphylococcal isolates tested contained more copies of IS256 than those associated with Tn4001 carried by these isolates. Three distinct copies of IS256 together with their flanking DNA were isolated by cloning from an EcoRI digest of the cellular DNA of the Gmr isolate, BM3121. These three copies of IS256 were shown to be intact. The results from DNA sequencing revealed that one of the three copies is flanked by 8-bp direct repeats, and it is suggested that this may be the result of insertion of the IS256 at this site by autonomous movement of the IS element. The insert DNA of all three clones hybridized to the cellular DNA prepared from a Staphylococcus aureus strain that carries neither IS256 nor the aacA-aphD gene. Two of the clones hybridized to several EcoRI fragments of the cellular DNA of this strain, indicating that in these cases IS256 is flanked by DNA present as several copies on the chromosome.     

Cloning of a human alpha(1,3)-fucosyltransferase gene that encodes ELFT but does not confer ELAM-1 recognition on Chinese hamster ovary cell transfectants.

In previous studies, Chinese hamster ovary (CHO) cell genomic DNA transfectants that expressed a human alpha(1,3)-fucosyltransferase (alpha(1,3)Fuc-T) gene were isolated and shown to possess a common approximately 7.5-kilobase (kb) EcoRI fragment that hybridized to an Alu probe (Potvin, B., Kumar, R., Howard, D. R., and Stanley, P. (1990) J. Biol. Chem. 265, 1615-1622). One of these transfectants was used to make a genomic DNA library in lambda ZAP-II from EcoRI-digested, size-selected (6-8 kb) DNA, and plaques that hybridized to an Alu probe were purified. After in vivo excision, two plasmids with DNA inserts greater than or equal to 6 kb were obtained and one of these (D2.1) conferred human alpha(1,3)-Fuc-T activity on CHO transfectants. A partial restriction map of this clone revealed an approximately 3.6-kb PstI fragment that contained an Alu sequence. This fragment was subcloned into pGEM-3Zf(+) and compared by restriction analyses with a previously described approximately 3.6-kb PstI DNA fragment isolated from a human peripheral blood lymphocyte library and shown to encode an alpha(1,3)-Fuc-T gene (Lowe, J. B., Stoolman, L. M., Nair, R. P., Larsen, R. D., Berhend, T. L., and Marks, R. M. (1990) Cell 63, 475-484). Both approximately 3.6-kb fragments gave identical restriction patterns. In addition, they both caused CHO transfectants to synthesize the Lex determinant Gal beta(1,4)[Fuc alpha(1,3)]GlcNAc beta 1 but not the alpha(2,3)-sialyl-Lex determinant. As expected, these transfectants did not bind to ELAM-1 on activated endothelial cells, since sialyl-Lex is a carbohydrate ligand recognized by ELAM-1. Surprisingly, however, an open reading frame encoded within the approximately 3.6-kb PstI fragment had a sequence identical to that of ELFT, an alpha(1,3)-Fuc-T previously reported to confer ELAM-1 binding on a previously reported to confer ELAM-1 binding on a CHO transfectant (Goelz, S. E., Hession, C., Goff, D., Griffiths, B., Tizard, R., Newman, B., Chi-Rosso, G., and Lobb, R., (1990) Cell 63, 1349-1356). Possible explanations for these apparently disparate results are discussed.     

EcoRI- and BamHI-families of repeated sequences in mustelids

The restriction enzymes EcoRI and BamHI digest the genomic DNAs from six mustelids species Mustela lutreola, M. vision, M. erminea, M. sibirica, Vormela peregusna, producing repeated fragments varying in length. Some fragments were hybridized to chromosomes and restriction digests of DNAs from some mustelids and other mammals. The 0.7 kb EcoRI repeats from DNA of M. erminea are dispersed over chromosomes of carnivors. The 1.35, 1.9 and 2.7 kb BamHI repeats from DNA of polecat M. putorius furo are specific for mustelids. These repeats demonstrate interspecific variation in length and the number of copies. All BamHI repeats have no strict tandem organization. The 1.9 kb BamHI repeats are concentrated in the heterochromatic pericentromeric regions and additional chromosome arms. The 1.35 kb BamHI repeats are only located in the centromeric regions of chromosomes of five species and are absent in Vormela peregusna.     

A partial sequence of lipoxygenase gene from genomic DNA of aromatic rice (Oryza sativa L.)

Aromatic (Bas-370, PB-1) and non-aromatic (Pusa-677, Pusa-834) rice were selected for the characterization and for distribution of lipoxygenase (Lox) genes. Polymorphism was observed when genomic DNA of rice varieties was hybridized with a heterologous lipoxygenase probe. A distinct polymorphic fragment (approximately 1.2 kb) was found in Bas-370. Sub-genomic library of Bas-370 was constructed and screened with LoxA probe. The smallest putative clone (pBas-14) of approximately 1.2 kb was sequenced. Complete nucleotide and deduced amino acid sequence showed the clone was 1134 bp long and comprised of 378 amino acid residues. PCR amplification of genomic DNA from four rice varieties with a soybean Lox primer also showed a polymorphic fragment of size approximately 600 bp (amplicon) in aromatic varieties that was sequenced directly. Nucleotide sequence alignment between pBas-14 and amplicon concluded that the amplicon was a part of the insert pBas-14.     

Characterization of proviruses cloned from mink cell focus-forming virus-infected cellular DNA

Two proviruses were cloned from EcoRI-digested DNA extracted from mink cells chronically infected with AKR mink cell focus-forming (MCF) 247 murine leukemia virus (MuLV), using a lambda phage host vector system. One cloned MuLV DNA fragment (designated MCF 1) contained sequences extending 6.8 kilobases from an EcoRI restriction site in the 5' long terminal repeat (LTR) to an EcoRI site located in the envelope (env) region and was indistinguishable by restriction endonuclease mapping for 5.1 kilobases (except for the EcoRI site in the LTR) from the 5' end of AKR ecotropic proviral DNA. The DNA segment extending from 5.1 to 6.8 kilobases contained several restriction sites that were not present in the AKR ecotropic provirus. A 0.5-kilobase DNA segment located at the 3' end of MCF 1 DNA contained sequences which hybridized to a xenotropic env-specific DNA probe but not to labeled ecotropic env-specific DNA. This dual character of MCF 1 proviral DNA was also confirmed by analyzing heteroduplex molecules by electron microscopy. The second cloned proviral DNA (designated MCF 2) was a 6.9-kilobase EcoRI DNA fragment which contained LTR sequences at each end and a 2.0-kilobase deletion encompassing most of the env region. The MCF 2 proviral DNA proved to be a useful reagent for detecting LTRs electron microscopically due to the presence of nonoverlapping, terminally located LTR sequences which effected its circularization with DNAs containing homologous LTR sequences. Nucleotide sequence analysis demonstrated the presence of a 104-base-pair direct repeat in the LTR of MCF 2 DNA. In contrast, only a single copy of the reiterated component of the direct repeat was present in MCF 1 DNA.     

Cloning and characterization of the gene for the yeast cytoplasmic threonyl-tRNA synthetase.

A fragment of DNA from the yeast nuclear gene MST1 that codes for the mitochondrial tRNAThr1 synthetase was used as a probe to screen for other yeast threonyl-tRNA synthetase genes. At low stringency, the MST1 probe hybridizes strongly to a 6.6 kb EcoRI fragment of yeast genomic DNA with the homologous gene and in addition hybridizes more weakly to a smaller 3.6 kb EcoRI fragment with a second threonyl-tRNA synthetase gene (THS1). To clone THS1, a library was constructed by ligation to pUC18 of size selected (3-4.5 kb) EcoRI fragments of genomic DNA. Several clones containing the 3.6 kb EcoRI fragment were isolated. A 2,202 nucleotide long open reading frame corresponding to THS1 has been identified in the cloned fragment of DNA. The predicted protein encoded by THS1 is 38% identical to the E. coli threonyl-tRNA synthetase over the latter's length (642 amino acids) and is 42% identical to the predicted MST1 product over its 462 residues. In situ disruption of the chromosomal copy of THS1 is lethal to the cell, indicating that this gene codes for the cytoplasmic threonyl-tRNA synthetase.     

Interspersion of mouse satellite deoxyribonucleic acid sequences

DNA sequences with homology to the major (A + T)-rich mouse satellite component were localized in CsCl gradients by hybridization with a labeled satellite cRNA probe. Although, as expected, most of the hybridization was to DNA in the satellite-rich shoulder, substantial radioactive cRNA hybridized with DNA from denser regions of the gradient. Further examination revealed that hybridization to main-band DNA was not due to physical trapping of satellite DNA in the gradient, and melting experiments argue that the associated radioactivity was due to true RNA/DNA hybridization. Nearest-neighbor analysis of hybridized [alpha-32P]CTP-labeled l-strand cRNA indicates that hybridization to main-band DNA is by the satellite cRNA and not a contaminant. Together, these data argue that mouse satellite-like sequences are interspersed within the main-band fraction of DNA. For the support of this contention, total mouse DNA, purified main-band DNA, and purified satellite DNA were digested with EcoRI, sedimented in a sucrose gradient, and hybridized with labeled satellite cRNA. Mouse satellite DNA is not cleaved with EcoRI, so that purified EcoRI-digested satellite DNA sediments as a high molecular weight component. When total mouse DNA is digested with EcoRI, the majority of satellite-like sequences remain as high molecular weight DNA; however, significant amounts of satellite-like sequences sediment with the bulk of the lower molecular weight digested DNA, lending further credence to the argument that satellite-like sequences are interspersed with main-band DNA.     

Replication of the Streptomyces plasmid pSN22 through single-stranded intermediates

The replication of the 11 kb conjugative multicopy Streptomyces plasmid pSN22 was analyzed. Mutation and complementation analyses indicated that the minimal region essential for plasmid replication was located on a 1.9 kb fragment of pSN22, containing a trans-acting element encoding a replication protein and a cis-acting sequence acting as a replication origin. Southern hybridization showed that minimal replicon plasmids accumulated much more single-stranded plasmid molecules than did wild-type pSN22. Only one strand was accumulated. A 500 by fragment from the pSN22 transfer region was identified which reduced the relative amount of single-stranded DNA, when added in the native orientation to minimal replicon plasmids. This 500 by DNA sequence may be an origin for second-strand synthesis. It had no effect on the efficiency of co-transformation, plasmid incompatibility, or stability. The results indicate that pSN22 replicates via single-stranded intermediates by a rolling circle mechanism.     

Replication of the Streptomyces plasmid pSN22 through single-stranded intermediates

The replication of the 11 kb conjugative multicopy Streptomyces plasmid pSN22 was analyzed. Mutation and complementation analyses indicated that the minimal region essential for plasmid replication was located on a 1.9 kb fragment of pSN22, containing a trans-acting element encoding a replication protein and a cis-acting sequence acting as a replication origin. Southern hybridization showed that minimal replicon plasmids accumulated much more single-stranded plasmid molecules than did wild-type pSN22. Only one strand was accumulated. A 500 by fragment from the pSN22 transfer region was identified which reduced the relative amount of single-stranded DNA, when added in the native orientation to minimal replicon plasmids. This 500 by DNA sequence may be an origin for second-strand synthesis. It had no effect on the efficiency of co-transformation, plasmid incompatibility, or stability. The results indicate that pSN22 replicates via single-stranded intermediates by a rolling circle mechanism.     

A single oligonucleotide can be used to rapidly isolate DNA sequences flanking a transposon Tn5 insertion by the polymerase chain reaction.

We have developed a strategy to rapidly construct DNA hybridization probes for the isolation of genes disrupted by transposon Tn5 insertions. A single oligonucleotide complementary to and extending outward from the ends of the inverted repeat of Tn5 was used to prime DNA synthesis in the polymerase chain reaction. The amplified product consisted of DNA sequences adjacent to both ends of the transposon insertion. The general feasibility of the approach was tested by amplifying pBR322 sequences from a derivative of pBR322 containing a Tn5 insertion. To amplify genomic DNA sequences flanking a Tn5 insertion in the chromosome of a Pseudomonas syringae strain, circular substrates were generated by ligating EcoRI-digested genomic DNA. Tn5 was contained intact within one such circular molecule, as the transposon does not contain sites for cleavage by EcoRI. The amplified product (approximately 2.5 kb) was used as a DNA hybridization probe to isolate the homologous fragment from a cosmid library of wild-type Pseudomonas syringae genomic DNA. This approach may be applied to the efficient isolation of sequences flanking any Tn5 insertion.     

Novel germline hMSH2 genomic deletion and somatic hMSH2 mutations in a hereditary nonpolyposis colorectal cancer family

Germline and somatic mutations of the hMSH2 gene were determined in a Japanese hereditary nonpolyposis colorectal cancer (HNPCC) family fulfilling the Amsterdam criteria. PCR-SSCP-sequencing of genomic DNA detected a somatic hMSH2 mutation of an A deletion at codon 227-229 in a duodenal carcinoma and a somatic hMSH2 mutation of an A insertion at codon 21 in a gastric carcinoma from affected family members, both carcinomas exhibiting high microsatellite instability. However, no germline hMSH2 mutation was detected by the PCR-SSCP-sequencing method. Genomic DNA was then analyzed by Southern blot hybridization using three hMSH2 cDNA probes (probe A involving exons 1-5, probe B involving exons 4-11 and probe C involving exons 9-16) after digestion by restriction enzymes, EcoRI, HindIII and NsiI. The NsiI digest of DNA from normal tissues of affected members exhibited an aberrant 8.6 kb restriction fragment, in addition to the normal 10.6 kb fragment, when hybridized to probes A and B. This suggested the presence of a heterozygous 2kb genomic deletion encompassing exon 4, 5 or 6. RT-PCR-sequencing revealed that the deleted region encompassed exon 5. This novel genomic deletion of the hMSH2 gene was confirmed to be pathogenic, and the Southern hybridization pattern was applied to the pre-symptomatic diagnosis.     

Genomic representation of the Hind II 1.9 kb repeated DNA.

The genomic representation and organization of sequences homologous to a cloned Hind III 1.9 kb repeated DNA fragment were studied. Approximately 80% of homologous repeated DNA was contained in a genomic Hind III cleavage band of 1.9 kb. Double digestion studies indicated that the genomic family, in the majority, followed the arrangement of the sequenced clone, with minor restriction cleavage variations compatible with a few base changes. Common restriction sites external to the 1.9 kb sequence were mapped, and hybridization of segments of the cloned sequence indicated the 1.9 kb DNA was itself not tandemly repeated. Kpn I bands which were homologous to the sequence contained specific regions of the repeat, and the molecular weight of these larger fragments could be simply explained. Mapping of common external restriction sites indicated that in some but not all cases the repeat could be organized in larger defined blocks of greater than or equal to 5.5 kb. In some instances, flanking regions adjacent to the repeat may contain common DNA elements such as other repeated DNA sequences, or possibly rearranged segments of the 1.9 kb sequence. It is suggested that although the 1.9 kb sequence is not strictly contiguous, at least some of these repeated sequences in the human genome are arranged in clustered or intercalary arrays. A region of the 1.9 kb sequence hybridized to a mouse repeated DNA, indicating homology beyond the primates.     

Specific DNA probe for detecting Legionella pneumophila

A fragment of the gene for cytolysin has been cloned. The product of the gene has been earlier identified as an immunoserological marker of Legionella pneumophila. Clones were selected by immunodetection of cytolysin gene product expression. An EcoRI 3.8 kb fragment of the genomic DNA from Legionella pneumophila strain Philadelphia-1 was used as a DNA probe that hybridized with the bacterial colonies of 20 Legionella pneumophila strains but not with the colonies of 10 other Legionella species or 8 other bacterial genera. The cloned fragment has been shown to be unique for Legionella pneumophila. The region of homology is suggested to be longer than a possible dimension of the cytolysin gene.