Characterization of a cloned chromosomal fragment affecting the proteinase activity of Streptococcus lactis ssp. lactis

Plasmid pVS8 (14.3 kbp) contains a 9.3 kbp fragment of Streptococcus lactis ssp. lactis SSL135 chromosomal DNA associated with the ability of this strain to grow in milk. In this study, it was found that pVS8 complements a defective plasmid-linked proteinase gene of SSL135. Using deletions and insertions, it was found that the size of the complementing region of pVS8 is approximately 6.0 kbp, and that its main part is located within a 5.7 kbp BglII fragment.     

Cellular transformation by subgenomic feline sarcoma virus DNA

The genome of the Snyder-Theilen strain of feline sarcoma virus (ST-FeSV) is a 4.3-kilobase-pair (kbp) RNA molecule that contains a 1.5-kbp cellular insertion (fes gene) flanked by feline leukemia virus sequences at its 5' end (1.6 kbp) and 3' end (1.2 kbp) (Sherr et al., J. Virol. 34:200-212, 1980). DNA transfection techniques have been utilized to determine the regions of the ST-FeSV genome involved in malignant transformation. I have found that the 3.7-kbp 5'-end fragment of the ST-FeSV provirus (which corresponds to the 3.4-kbp 5'-end fragment of the viral genome) is sufficient to transform NIH/3T3 fibroblasts. Enzymes that cleave the ST-FeSV provirus DNA within the feline leukemia virus gag gene sequences or within the fes gene abolished the transforming activity. Preservation of the proviral large terminal repeats was also required for transformation. Transformed NIH/3T3 cells obtained by transfection of total or subgenomic ST-FeSV DNA expressed normal levels of the ST-FeSV gene product ST P85 and of its associated protein kinase activity. Furthermore, these cells contained high levels of phosphotyrosine residues, a biochemical marker associated with cellular transformation induced by certain retroviruses including ST-FeSV. These results, taken together, strongly support the concept that only those ST-FeSV proviral sequences necessary for ST P85 expression are involved in malignant transformation.     

T-DNA and opine synthetic loci in tumors incited by Agrobacterium tumefaciens A281 on soybean and alfalfa plants.

We report here the molecular characterization of transferred DNA (T-DNA) in leguminous tumors incited by Agrobacterium tumefaciens A281 harboring the tumor-inducing plasmid pTiBo542. The T-DNA is composed of two regions named TL (left portion)-DNA and TR (right portion)-DNA, in accordance with the nomenclature for the octopine strains. TL-DNA is defined by several internal HindIII restriction fragments totaling 10.8 kilobase pairs (kbp) in uncloned soybean and alfalfa tumors. Alfalfa tumor DNA may contain one more HindIII fragment at the left end of TL-DNA than does soybean tumor DNA. TR-DNA has a 5.8-kbp BamHI-EcoRI internal fragment. All borders other than the left border of TL-DNA appear to be the same within the detection limits of Southern blot hybridization experiments. The two T-DNA regions are separated by 16 to 19 kbp of DNA not stably maintained in tumors. The distance from the left border of TL-DNA to the right border of TR-DNA is approximately 40 kbp. Loci for the mannityl opines are situated in TR-DNA, based on genetic and biochemical criteria.     

Molecular cloning of an ice nucleation gene from Erwinia ananas and its expression in Escherichia coli

Abstract An approximately 7 kbp genomic DNA fragment was cloned from an ice nucleation-active (ina) strain of Erwinia ananas and defined as to its restriction enzyme site. When the DNA fragment was introduced into E. coli MM294, a potent ice nucleation activity was expressed. Both 0.7 kbp truncation from the 5'-end and 1.7 kbp truncation from the 3'-end were also effective in expressing the ice nucleation activity in E. coli . Therefore, the resulting DNA fragment of approximately 5 kbp was considered to be an ina gene and named ina A. It existed as a unique gene in this strain of E. ananas . No corresponding ina gene existed in an ice nucleation-inactive strain of E. milletiae .     

Restriction fragment length polymorphism of cardiac myosin heavy chain gene in rats and its strain distribution

The distribution of an RFLP in EcoRI fragments of the cardiac myosin heavy chain gene among 29 strains of laboratory rats was examined. Southern blot hybridization of rat genomic DNAs with rat cardiac myosin heavy chain cDNA as a probe demonstrated an interstrain variation in one of eight EcoRI fragments. Of the 28 inbred strains examined, 10 had a fragment of 10 kbp, whereas 18 had a fragment of 7.5 kbp. The 15 samples of the remaining strain (Iar: WI outbred stock) had fragments of either 7.5 kbp or 10 and 7.5 kbp, indicating that this strain has maintained heterogeneity of these fragments.     

5-Variable genome sequence of the LIVP vaccinia virus. A possible role for short direct repeats in the formation of deletions]

HindIII-O/N DNA fragments of vaccinia virus (VV) of the LIVP strain were mapped using thirteen restriction endonucleases. Nucleotide sequences of the HindIII-O fragment (1530 bp) as well as of a site of the HindIII-N genome fragment 353 bp in size were determined. Comparison of restriction maps and nucleotide sequences of VV strains (WR and LIVP) demonstrated that DNA of VV LIVP contained % deletions and 2 insertions. "Reliable" short direct repeats were localized and their possible role in formation of DNA deletions was shown. It was suggested that VV endonuclease and DNA-ligase participate in replication and repair processes. Mechanism of formation of variable sequences of viral genomes is discussed.     

Localization of the genes coding for the 51 kDa PSII chlorophyll apoprotein,apocytochrome b6, the 65–70 kDa PSI chlorophyll apoproteins and the 44 kDa PSII chlorophyll apoprotein in pea chloroplast DNA

Summary DNA probes isolated from previously mapped spinach genes were used to locate 5 genes on pea ctDNA by heterologous hybridization. The genes mapped include psbC, psaA, psaB, psbB, and petB. PsbB and petB mapped to a 6.7 kbp XbaI DNA fragment adjacent to the petD gene. Northern probes from within the DNA which codes for psbB and petD hybridized to 6 RNAs ranging from 1.2 to 5.6 kbp. The psaA and psaB genes, which code for 65–70 kDa proteins of Photosystem I, were mapped to a 7.5 kbp. XbaI DNA fragment. A 5.8 kbp RNA is transcribed from the region which contains the psaA and psaB genes suggesting that these genes are co-transcribed. Finally the psbC gene which codes for a 44 kDa chlorophyll-protein of Photosystem II was mapped to a 12.3 kbp PstI DNA fragment. The pea psbC open reading frame overlaps the psbD coding sequence and this gene pair is within 3 kbp of the psaA-psaB genes. Overall, the organization of the 3 gene clusters analyzed in peas is similar to that reported for spinach.     

Cloning of the ethidium efflux gene from Escherichia coli

The gene specifying the ethidium efflux system of Escherichia coli has been cloned on a 3.2 kbp HindIII fragment and located on a 1.2 kbp fragment within this. Cross-resistance studies indicate that the system has a broad specificity for monovalent cations and the gene shows no hybridisation with similar genes found in Staphylococci.     

Highly immunogenic variant of attenuated vaccinia virus

The LIVPΔ6 strain of vaccinia virus (VACV) was created by genetic engineering on the basis of previously obtained attenuated 1421ABJCN strain by target deletion of the A35R gene encoding an inhibitor of antigen presentation by the major histocompatibility complex class II. 1421ABJCN is the LIVP strain of VACV with five inactivated virulence genes encoding hemagglutinin (A56R), γ-interferon-binding protein (B8R), thymidine kinase (J2R), complement-binding protein (C3L), and Bcl2-like inhibitor of apoptosis (N1L). The highly immunogenic LIVPΔ6 strain could be an efficient fourth-generation attenuated vaccine against smallpox and other orthopoxvirus infections.     

Cloning of the outer membrane high-affinity Fe(III)-pyochelin receptor of Pseudomonas aeruginosa.

Pseudomonas aeruginosa produces the phenolic siderophore pyochelin under iron-limiting conditions. In this study, an Fe(III)-pyochelin transport-negative (Fpt-) strain, IA613, was isolated and characterized. 55Fe(III)-pyochelin transport assays determined that no Fe(III)-pyochelin associated with the Fpt- IA613 cells while a significant amount associated with KCN-poisoned Fpt+ cells. A P. aeruginosa genomic library was constructed in the IncP cosmid pLAFR1. The genomic library was mobilized into IA613, and a recombinant cosmid, pCC41, which complemented the Fpt- phenotype of IA613, was isolated. pCC41 contained a 28-kb insert of P. aeruginosa DNA, and the Fpt(-)-complementing region was localized to a 3.6-kb BamHI-EcoRI fragment by deletion and subcloning of the insert. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of IA613 revealed that it lacked a 75-kDa outer membrane protein present in Fpt+ strains. IA613 strains bearing plasmid pRML303, which carries the 3.6-kb BamHI-EcoRI fragment of pCC41, expressed the 75-kDa outer membrane protein and demonstrated a 55Fe(III)-pyochelin transport phenotype identical to that of a wild-type Fpt+ strain. Minicell analysis demonstrated that the 3.6-kb BamHI-EcoRI fragment of pCC41 encoded a protein of approximately 75 kDa. The results presented here and in a previous report (D. E. Heinrichs, L. Young, and K. Poole, Infect. Immun. 59:3680-3684, 1991) lead to the conclusion that the 75-kDa outer membrane protein is the high-affinity receptor for Fe(III)-pyochelin in P. aeruginosa.     

Unlike in other monocotyledonous plants such as wheat and rice, the region containing the tRNA (UCC), tRNA (UCU) and CF1 ATPase α-subunit genes has not undergone recombination in the leek chloroplast DNA

The nucleotide sequence of a 1.1 kbp BamHI fragment of the leek chloroplast DNA (Allium porrum., fam. Liliaceae) has been determined. The fragment contains the 3' part of the tRNA^Gly (UCC) gene and the tRNA^Arg (UCU) gene on the same strand, and the 3' end of the atpA gene encoding the CF₁ ATPase α-subunit which is located on the opposite strand. The gene arrangement and nucleotide sequence of this fragment are similar to those of the corresponding region in the tobacco chloroplast DNA but differ significantly from what has been observed in other monocotyledonous plants such as wheat and rice, in which the region containing these genes has undergone intensive rearrangement.     

Isolation of the tyrosine phenol lyase gene from Citrobacter freundii

Summary The gene for the enzyme tyrosine phenol-lyase (TPL) was initially isolated on a 45 kbp fragment of Citrobacter freundii genomic DNA contained in a cosmid. Subsequent restriction enzyme digestion and sub-cloning resulted in the gene being contained on a 2.4 kbp DNA fragment.     

A physical map of wheat chloroplast DNA showing the location of the structural genes for the ribosomal RNAs and the large subunit of ribulose 1,5-bisphosphate carboxylase

Summary The restriction endonucleases SalGI and PstI have been used to construct a physical map of wheat ctDNA. The molecule was found to contain approximately 135 kbq, and in common with many other higher plant ctDNAs about 15% of the sequences are repeated in an inverted orientation. It was established by electron microscopy that, in wheat, each segment of the inverted repeat contains 21.0 kbp, and that the single copy regions separating the two repeated segments contain 12.8 kbp and 80.2 kbp. Blot hybridisation showed that one set of ribosomal genes is located in each segment of the inverted repeat region and the sizes of these genes were accurately determined by measuring the dimensions of hybrids between the chloroplast rRNAs and the identified Sal and Eco fragments on electron micrographs: the genes for the 16S and 23S rRNAs contain 1530 bp and 2850 bp respectively and are separated by a spacer region of 2350 bp. The Bgl fragment of maize ctDNA known to contain the structural gene for the large-subunit (LS) of ribulose 1,5-bisphosphate carboxylase was used as a probe to locate the LS gene in wheat ctDNA. A small (2.8 kbp) Eco fragment was found to contain most of the wheat LS gene and is derived from the larger single-copy region, 23.5 kbp away from one segment of the inverted repeat and 54.8 kbp from the other.     

Cloning of the ADE2 gene of Saccharomyces cerevisiae and localization of the ARS-sequence

Mutational changes in ADE2 result in the accumulation of red pigment in cells, which serves as an indicator for the selection of mutants. This easily detectable phenotype of red-coloured colonies can account for the wide use of ade2 mutants in yeast genetics. ADE2 gene was cloned in a shuttle vector by complementing the ade2 mutation in the yeast. It was shown that the 2.2 kbp HindIII fragment of yeast DNA contains structural sequences of the ADE2 gene as well as the ARS sequence. Deletion analysis of the 5' end of the ADE2 gene showed the ARS sequence to be situated at the distal end of the 1 kbp HindIII fragment. Removal of the ARS sequence does not influence ADE2 gene complementation ability. Transformants containing the ADE2 gene comprised in their plasmids form white colonies. Loss of the plasmids results in colour change of colonies.