DNA Restriction Enzymes

A modified gel electrophoresis technique provided a reproducible way of detecting and isolating plasmids with molecular weights ranging from 12 X 10(6) to 370 X 10(6) for Azospirillum species. Analysis with the nifHD region of Rhizobium trifolii showed that the Azospirillum nif genes were chromosomally located in all eight strains investigated and not on endogenous plasmids.

As an approach to the study of mammalian gene expression, the promoters and translation initiation regions of the rat preproinsulin II and the simian virus 40 early genes were fused to the structural gene of Escherichia coli 3-galactosidase, a sensitive probe for gene expression. These fusions were introduced into COS-7 cells, a simian virus 40 large tumor-antigen-producing monkey kidney cell line, where they directed the synthesis of enzymatically active hybrid 3-galactosidase proteins. Conditions for transfection were varied to optimize the expression of 13-galactosidase activity in the transfected cells. The pH optimum of this activity was found to be 7.0, the same as that of native E. coli fgalactosidase and distinct from the major lysosomal "acid" fl-galactosidase. The fused preproinsulin-fl-galactosidase was further characterized by gel electrophoresis of nondenatured cell extracts stained by a fluorogenic substrate and by immunoprecipitation and gel electrophoresis of 3H-labeled cell proteins. These results all indicate that fully active tetrameric P-galactosidase hybrids can be produced in mammalian cells. The expression of preproinsulin-,B-galactosidase activity was measured in the presence of high glucose, insulin, dexamethasone, or epidermal growth factor but no regulatory changes were observed. Gene fusions utilizing the Escherichia coli lacZ structural gene provide an especially suitable method for studying gene expression and regulation (1). Its product, f3-galactosidase, can be as

Several DNA fragments carrying tRNA genes have been cloned from EcoRI endonuclease digests of Escherichia coZi DNA. Using cloned DNA, the sequence of the region around the distal gene for tRNAlASP(F(or G)) in the E. coZi ribosomal RNA operon [rrnF(or G)] has been determined. In the distal portion of rrnF(or G), the genes for 23S, 5S rRNA and tRN41P(F(or G)) are located in that order and separated by intergenic spacers of 93 and 52 base pairs, respectively. A possible hairpin structure, with its center between the 22nd and 23rd base pair downstream from the 3'-end of the tRNAAsP(F(or G)) gene, followed by a sequence of eight thymidine residues was identified as the transcription termination signal for rrnF(or G). The termination is rhoindependent, at least in vitro, and occurs within the region of the contiguous thymidine residues. A possible promoter for a protein gene is present about 50 base pairs downstream from the rrnF(or G) terminator.

We have utilized 2 recombinant DNA strategies for immunization against FeLV in cats: (a) modified live virus was attenuated by mutation and recombination, and (b) an immunogen, consisting of subunit envelope protein, was prepared in genetically engineered yeast. Results indicated that the genetically manipulated live virus preparations were not protective against FeLV challenge because they were either not attenuated in virulence or were not sufficiently antigenic. Immunization with yeast-synthesized FeLV envelope protein followed by the modified live virus gave protective immunity in cats under experimental conditions. Future immunization attempts will concentrate on enhancing the immunogenic potency of the yeast- synthesized FeLV envelope protein.

Several bacteriological surveys were performed from 1994 to 1996 at different Litopenaeus vannamei hatcheries (in Ecuador) and shrimp farms (in Mexico). Samples were taken from routine productions of healthy and diseased L. vannamei larvae, postlarvae, and their culture environment and from healthy and diseased juveniles and broodstock. In Ecuador, the dominant bacterial flora associated with shrimp larvae showing symptoms of zoea 2 syndrome, mysis mold syndrome, and bolitas syndrome has been determined. Strains were characterized by Biolog metabolic fingerprinting and identified by comparison to a database of 850 Vibrio type and reference strains. A selection of strains was further genotypically fine typed by AFLP. Vibrio alginolyticus is predominantly present in all larval stages and is associated with healthy nauplius and zoea stages. AFLP genetic fingerprinting shows high genetic heterogeneity amongV. alginolyticus strains, and the results suggest that putative probiotic and pat...

A group of cosmid clones was isolated from the region of the mouse t complex and analysed by a rapid restriction mapping protocol based on linearization of circular cosmid DNA in vitro. A plasmid capable of producing high levels of phage J terminase was constructed and procedures for in vitro cleavage of cosmid DNAs were optimised. After linearization, the cosmids were partially digested'with restriction enzymes, and either cos end was labelled by hybridization with radioactive oligos complementary to the cohesive end sequence, a step which we have described previously for clones in phage I (Rackwitz et al., 1984). High-resolution restriction maps derived by this method were used to identify and align the cosmids, to localise the position of repetitive sequences, and to interpret the results of electron microscopy heteroduplex experiments.

A human galactoside-binding protein with an M, of 31,000 was cloned from the human III-1(180 fibrosarcoma complementary DNA library. A partial complementary DNA clone containing the complete coding region was characterized and the deduced sequence encodes a polypeptide of 242 amino acids with the characteristics of a carbohydrate-binding pro tein. The gene coding for the human galactoside-binding protein was mapped to the chromosomal band Ipl3. The deduced amino acid sequence of the human galactoside-binding protein revealed 95 residues at the amino terminus, homologous to the predicted amino acid sequence of the second exon of the human l.-myc gene.

From a library of mouse sperm DNA, we have isolated two overlapping clones which contain the Cc, gene. One of these clones also contains the C,6 gene. The C gene is separated from the C membrane exons by approximately 2 kilobases (kb) of DNA. Ae Cc, gene was identified by (a) hybridization to poly(A) RNA prepared from the IgD-producing rat plasma cell tumor IR731, and (b) homology of a translated nucleotide sequence to the amino acid sequence of the human 8 chain. The C gene spans 8 kb of DNA in the germ line. Plasmid subclones of the Cc, gene were used as probes in Southern and RNA blot experiments. RNA blot analysis of cytoplasmic poly(A)+RNA from 1R731 and a piA+8 B-cell hybridoma revealed 1.6-and 2.7-kb 8 mRNA species with different 3' ends, which presumably encode the secreted and membrane-bound forms, respectively, of the 8 chain. Southern blot analysis of DNA from two ,u+8+ lymphomas revealed that the C5g gene is in the germ-line configuration in each case. Restriction map analysis of C and Cc, genomic clones isolated from a library of normal IA+ 8B-cell DNA also gave no evidence for DNA rearrangement in the region between the C, and Cog genes. Taken together, these data suggest that IgD expression in pI+B+ B cells does not involve a VH-to-Cs DNA switch rearrangement. We propose that simultaneous expression of CA and Cc with a single V gene is mediated by two alternative routes of RNA processing o? a primary nuclear transcript which contains the VH, C , and Cc, genes. In contrast, analogous experiments with myeloma TR731 DNA revealed that the C,, gene has been deleted from the myeloma DNA and that the Cog gene has undergone DNA rearrangement, presumably including a switch recombination of the VH gene from the C to the Cc, gene. These results indicate that two alternative meclanisms may be used in the expression of IgD molecules-RNA splicing in B cells and DNA rearrangement in plasma cells. An immunoglobulin heavy chain is composed of a variable (VH) region and one of five classes of constant (CH) region: CG (gM),

We have used blot hybridization techniques to analyze transcription and repetitive DNA sequence distribution in the chicken f-like globin gene region. Various cloned DNAs, spanning the fi-globin genomic region, were digested with restriction enzymes, electrophoresed on agarose gels, and blotted to nitrocellulose. The blots were hybridized to either nick-translated DNA or pulse-labeled RNA transcribed in vitro from the nuclei of 5-or 12-day chick embryo red blood cells. Analysis of the results has led to the following conclusions. 1) The hatching gene, fH, identified in previous work based solely on its homology to fi-globin cDNA, is a transcriptionally active gene which is transcribed in the red cell nuclei of 12-day but not 5-day embryos. 2) Each of the four f-like globin genes (p, pfH, , e) is transcribed as an independent transcription unit. 3) The adult gene, , may have two well separated promoters. 4) There is a new, developmentally regulated transcription unit in the f-globin region, closely linked to the fi-globin genes. 5) The embryonic globin genes, e and p, are embedded in clusters of repeat sequences. 6) Some of the repeats recur in the different clusters but are in scrambled order and are interspersed with segments of unique sequence DNA. 7) Intermingled in each cluster are members of repetitive DNA families characterized by vastly different repeat frequencies and transcriptional levels. The most actively transcribed repeat has many of the characteristics of the human Alu family. Some repeats are transcriptionally silent. 8) At least two repeat sequences are members of families whose transcription is developmentally regulated. These two repeats lie in the 3' flanking domain of the E gene. The repeat families they represent are regulated coordinately with the e gene but are transcribed separately from it and at a much higher level, presumably as segments of transcription units dispersed throughout the genome. Such repeats may serve to regulate the expression of the transcription units of which they are part. The complete chicken-like globin gene region has been cloned and mapped (Villeponteau and Martinson, 1981; Dolan

The two major protease inhibitors in mouse plasma are alpha 1-protease inhibitor (alpha 1-PI), putative inhibitor of neutrophil elastase, and contrapsin, an inhibitor in vitro of trypsinlike proteases. We have shown by nucleotide sequence analysis that these two inhibitors are related (R. E. Hill, P. H. Shaw, P. A. Boyd, H. Baumann, and N. D. Hastie, Nature (London) 311:175-177, 1984). Here, we show that the contrapsin and alpha 1-PI genes are members of two different multigene families, each containing at least three genes in mice and rats. We established the chromosomal locations of these genes by analyzing the segregation of restriction fragment length polymorphisms in recombinant inbred mouse strains. These experiments show that the multiple genes in each family are clustered and that the two gene families are closely linked on chromosome 12. Thus the genes for contrapsin and alpha 1-PI are likely to have evolved by duplication of a common ancestral gene. The contrapsin multigen...

We have combined three different methods, deletion mapping of T-cell lines, field-inversion gel electrophoresis, and the restriction mapping of a cosmid clone, to construct a physical map of the murine T-cell receptor flchain gene family. We have mapped 19 variable (Va) gene segments and the two clusters of diversity (Do) and joining (p) gene segments and constant (Cp) genes. These members of the fl-chain gene family span %450 kilobases of DNA, excluding one potential gap in the DNA fragment alignments. The antigen-binding T-cell receptors are encoded in mice by two families of genes-the a-chain gene located on murine chromosome 14 and the p-chain genes on chromosome 6 (1-5). The p chains are encoded by a constant gene (C,3) and variable (1,9), diversity (D.8), and joining (4) gene segments, which rearrange and are joined together at the DNA level during T-cell differentiation to generate an assembled Vat gene (6-10). The ,-chain gene family of murine T-cell receptors contains just 20-30 Vat gene segments by statistical estimates (11, 12), 19 of which have been identified from cDNA or genomic clones (13, 14). This family also encodes two closely linked D,3-J,3-C,3 clusters, each containing 1 D, and 7 J,3 gene segments as well as a single C,3 gene (6-10). One V1, gene segment, V,314, is located 10 kilobases (kb) downstream from the C,2 gene (15). The relative order and locations of the 18 other V1, gene segments are unknown, although it is generally thought that they are encoded within hundreds of kilobases of DNA 5' to the D,3-J,3-C,3 gene clusters. We have employed three distinct, but complementary, approaches for mapping the 8-chain gene family. First, deletional mapping has permitted us to exploit the deletional rearrangement mechanisms of most T cells. On a chromosome that has experienced a V,-D,-4p deletional rearrangement, the V,3 gene segments located between the rearranging Vp and Dp gene segments are lost. The identity of the

DNA can be sequenced by a chemical procedure that breaks a terminally laleled DNA molecule partially at each repetition of a base. The lengths of the labeled fragments then identify thepositions of thatse. We describe reactions that cleave DNA Preferentially at guanines, at adenines, at cy

The purpose of this study was to determine aspects of the epidemiology of nosocomial infection due to Candida parapailosds. Can-&da specie43 are important nosocomial pathogenq however, little epidemiologic information is available. PATIENTS AND =ODSz We prospectively cul-turtxlspecimensfrom98patientsadmittedtothe bonemarrowkanspbmtunitandamedkiueintensivecareunit(ICU)ofatertkycarehospital. &&mensfromhandsofpersonnelandenviron-msncal~W~also-~-tal cxlhalmWere~befOrepe~~Were&dmitted toastudieduniLRe+Wk&m~analysis (RZA)ofchromawwlalDNAwasusedasatyp~ qWt4!!mtodeWmhietherela~of~ RESULTS C. parap&o&was idenMed from five patif3ut8$sixhandl?ultWsfromfour~staff, andtwoen-tall3u&ce%Allfivepatients hadIMgatlvehlitkllc&lllWl#UdaCquhX!dCjW ap?3&J&aft%radmis&mtothestudyunitaThere wereno&ni&xmtdiffer0ncesbelxveenpatien~ and~tE-olsUbje@Jinage,~diclegse,im-m~therapy,andh&nunentation The duration of autibiotic therapy (mediam 328 velw311J3days#p=OS06)andthedurationinthe unit(me4~30.1ve~3us16.1days,p=0.048)was longer in patlenta than in controls. No common sourcewasiden~REAlwealedthreestrahl typee;-,Oll0f3tdltypewaSideolticalh fourpatlentS#threestaffmemberi%andtwoenvironmental-CONCLUSION: Them redts suggest exogenous acquisition of C. pa.ra@osk Based upon isolation of identical patient straius of C. parap~&~& from inanimate surf&es before patients were admitted to a study unit, there is evidence that the organism may have been acquired from the hos-From the Departments of Internal Medicine (VS. JAV. LD. JDS. MJZ) and Biostatistics @B-J). Division of Infectious Disease (VS, JAV. LD, JDS, MJZ).

The gene encoding an aspartic proteinase precursor (proplasmepsin) from the rodent malaria parasite Plasmodium berghei has been cloned. Recombinant P. berghei plasmepsin hydrolysed a synthetic peptide substrate and this cleavage was prevented by the general aspartic proteinase inhibitor, isovaleryl pepstatin and by Ro40-4388, a lead compound for the inhibition of plasmepsins from the human malaria parasite Plasmodium falciparum. Southern blotting detected only one proplasmepsin gene in P. berghei. Two plasmepsins have previously been reported in P. falciparum. Here, we describe two further proplasmepsin genes from this species. The suitability of P. berghei as a model for the in vivo evaluation of plasmepsin inhibitors is discussed.

Haemophilia A is the most common inherited bleeding disorder in man, affecting approximately 1 male in 10,000. The disease is caused by a deficiency in the gene for factor VIII, a component of the intrinsic coagulation pathway. Due to the broad range of clotting activity in normal and heterozygous females, it is often difficult to confirm the status of women at risk for carrying the disease. A genetic marker in the form of a restriction fragment length polymorphism (RFLP) within or tightly linked to the factor VIII gene would serve as a tag for the haemophilia gene, thus allowing both accurate carrier detection and improved, earlier prenatal diagnosis by chorionic villi sampling. The recent isolation of the factor VIII gene has allowed a search for RFLPs within the gene, and we report here the identification of a common polymorphism within the factor VIII gene, revealed by the restriction enzyme BclI, which can be used diagnostically in about 42% of all families. Although the diseas...

The differentiation of Bifidobacterium species was performed with specific primers using the PCR technique, the amplified ribosomal DNA restriction analysis (ARDRA) technique based on reports on the sequence of the 16S rRNA gene and speciation based on a short region of the ldh gene. Four specific primer sets were developed for each of the Bifidobacterium species, B. animalis, B. infantis and B. longum. The use of the ARDRA method made it possible to discriminate between B. infantis, B. longum and B. animalis with the combination of BamHI, TaqI and Sau3AI restriction enzymes. The ldh gene sequences of 309^312 bp were determined for 19 Bifidobacterium strains. Alignment of these short regions of the ldh gene confirmed that it is possible to distinguish between B. longum and B. infantis but not between B. lactis and B. animalis. ß 2000 Published by Elsevier Science B.V. on behalf of the Federation of European Microbiological Societies.

We present a DNA-based implementation of reaction system with molecules encoding elements of the propositional logic, that is, propositions and formulas. The protocol can perform inference steps using, for example, modus ponens and modus tollens rules and de Morgan's laws. The set of the implemented operations allows for inference of formulas using the laws of natural deduction. The system can also detect whether a certain proposition a can be deduced from the basic facts and given rules. The whole protocol is fully autonomous; that is, after introducing the initial set of molecules, no human assistance is needed. Only one restriction enzyme is used throughout the inference process. Unlike some other similar implementations, our improved design allows representing simultaneously a fact a and its negation ~a, including special reactions to detect the inconsistency, that is, a simultaneous occurrence of a fact and its negation. An analysis of correctness, completeness, and complex...

SUMMARY Restriction endonuclease analysis of specific gene sequences is proving to be a valuable technique for characterisation and diagnosis of inherited disorders. This paper describes detailed protocols for isolation, restriction, and blot hybridisation of genomic DNA. ...

We present complete restriction endonuclease cleavage site maps of the bacteriophage P22 chromosome for 16 enzymes with six base recognition sequences, thereby positioning 116 new sites on the chromosome. Twenty-four such restriction maps for P22 DNA, containing 162 sites, have now been completed, and three enzymes were found that did not cut P22 DNA. Our results are consistent with the ideas that ClaI does not cleave the methylated recognition sequence ATCGA(me)T or A(me)TCGAT and StuI does not cleave the methylated recognition sequence AGGCC(me)T.

When a DNA fragment containing a marker gene was ligated to random chromosomal fragments of Streptococcus pneumoniae and used to transform a recipient strain lacking that gene, the gene was integrated at various locations in the chromosome. Such ectopic integration was demonstrated for the malM gene, and its molecular basis was analyzed with defined donor molecules consisting of ligated fragments containing the malM and sul genes of S. pneumoniae. In a recipient strain deleted in the mal region of its chromosome, these constructs gave Mal+ transformants in which the malM and sul genes were now linked, with malM located between duplicate sul segments. Ectopic integration was unstable under nonselective conditions; mal(sul) ectopic insertions were lost at a rate of 0.05% per generation. Several possible mechanisms of ectopic integration were examined. The donor molecule is most likely to be a circular form of ligated homologous and nonhomologous fragments that, after entry into the ce...

A nomenclature is described for restriction endonucleases, DNA methyltransferases, homing endonucleases and related genes and gene products. It provides explicit categories for the many different Type II enzymes now identi®ed and provides a system for naming the putative genes found by sequence analysis of microbial genomes.

Since the discovery of plasmid, various methods have been developed to isolate plasmid DNA. All the methods have one common and important target of isolating plasmid DNA of high quality and quantity in less time. These methods are not completely safe because of use of toxic chemicals compounds. The developed protocol for plasmid extraction is based on the alkaline lysis method of plasmid preparation (extraction atpH 8.0) with slight modifications. Cell lysis reagent sodium dodecyl sulfate is replaced by lipase enzyme present in laundry detergent. A good plasmid preparation can be made, which is well suited for subsequent molecular biology applications. By taking safety measures on count, contaminants like, RNA and protein can be completely avoided with maximized plasmid yield. The resultant plasmid quality and quantity can be well comparable to other prevalent methods.

Cystic fibrosis (CF) is the most common genetic disease among Caucasians. The CF gene, named cystic fibrosis transmembrane conductance regulator (CFTR), codifies a protein that acts as a channel through the epithelial membrane. The present work aimed (1) to detect sequence alterations in the nucleotide binding regions and at the membrane spanning domain of the CFTR gene and (2) to detect the following frequent mutations R347P, R347H, R334W, and Q359K (located in exon 7), DF508 (located in exon 10), G542X, G551D, R553X, and S549N (located in exon 11), W1282X (located in exon 20), and N1303K (located in exon 21). Seventyseven unrelated CF patients were analyzed, who were previously diagnosed and currently under treatment at the Pneumology Service of our hospital. Regions of interest were amplified by PCR using specific primers. Each sample was analyzed by a nonradioactive single-stranded conformational polymorphism (SSCP) analysis technique and restriction enzyme digestion. The DF508 mutation was found in 48.7% of the alleles. Frequencies of G542X, R334W, R553X, and W1282X mutations in our population were 3.25, 1.3, 0.65, and 0.65%, respectively. No alleles were found to carry mutations G551D, R334W, R347P, R347H, Q359K, S549N, and N1303K, which were included in the screening protocol. This study allowed the characterization of 84 out of 154 CF mutant alleles (54.5%). The incidence of main CF mutations analyzed was similar to that of the south European population. Mutation data presented here will be useful for designing new DNA testing strategies for CF in South Brazil.

SOURCE/DESCRIPTION: a 850 bp cDNA insert from a liver library constructed in the PstI site of pEXI. It covers from the aa 121 to the aa 396 of the plasmatic coagulation factor XII. POLYMORPHISM : a polymorphic TaqI band of 2.1 kb and two constant bands of 3.3 and 1.3 kb. FREQUENCY: studied in 45 unrelated Italian subjects. The polymorphism has been detected on'ly in one family with factor XII deficiency. NOT POLYMORPHIC FOR: Bam HI, BclI, BglII, PstI, HindIII, with DNA from 6 unrelated subjects. CHROMOSOMAL LOCALISATION: autosomic. MENDELIN INHERITANCE: co-dominant segregation shown in 1 family (see figure). PROBE AVAILABILITY: write to Marco Tripodi. OTHER COCvIENTS: wash stringency lxSSC 600. AcKtLEDGEENTS: this work was supported by the Progetto Finalizzato Ing.Gen. e Basi Mol. Mal.Ered. contratto CNR n.8400877.

Fixed mitotic chromosomes of A. tornosi have been analysed by means of C-banding, DA-DAPI and Chromomicin A3 fluorescence, as well as by digestion in situ with Alu I, Hae III, Hinf I and Hind III restriction endonucleases. From the results obtained at least nine types of chromatin can be distinguished in A. tornosi. Some C-band positive areas (constitutive heterochromatin) which show a characteristic fluorescence pattern are digested by specific endonucleases, whilst others are undigested. C-band negative areas (euchromatin) are digested by some restriction endonucleases but not by others. Regions digested are supposed to contain highly repetitive DNAs. It is noteworthy, however, that the heterochromatin associated with NORs is not attacked by any of the enzymes we used, while regions believed to contain AT-rich DNA (DA-DAPI positive) are digested by Hae Ill that cleaves the GGCC base sequence target.

Many different single nucleotide polymorphisms (SNPs) genotyping methods have been developed recently. However, most of them are expensive. Using restriction enzymes for SNP genotyping is a cost-effective method. However, restriction enzyme mining for SNPs in a genome sequence is still challenging for researchers who do not have a background in genomics and bioinformatics. In this review, the basic bioinformatics tools used for restriction enzyme mining for SNP genotyping are summarized and described. The objectives of this paper include: i) the introduction of SNPs, genotyping and PCR-restriction fragment length polymorphism (RFLP); ii) a review of components for genotyping software, including tools for primer design only or restriction enzyme mining only; iii) a review of software providing the flanking sequence for primer design; iv) recent advances in PCR-RFLP tools and natural and mutagenic PCR-RFLP; v) highlighting the strategy for restriction enzyme mining for SNP genotyping;...

The complete protein precursor of human kidney renin has been determined from the sequence of cloned genomic DNA. The gene spans 12 kilobases of DNA and is interrupted by eight intervening sequences. The nine regions (exons) encoding the protein were mapped with a mouse renin cDNA probe, synthetic oligonucleotide probes, and by hybridization of genomic restriction fragments to a 1600-nucleotide human kidney mRNA. The predicted 403-amino acid preprorenin consists of mature renin and a 66-residue amino-terminal prepropeptide. The DNA sequence 5' to the first exon indicates the location of a transcriptional promoter (T-AT A A A) for a mRNA encoding preprorenin. An additional transcriptional promoter site is located within the first intron, which, if used, would express a shortened nonsecreted prorenin. The structure of the human renin gene is similar to that of human pepsinogen, a closely related aspartyl protease enzyme. This observation suggests that renin and pepsinogen have a common evolutionary origin.

In partially purified preparations of the vacuolar ATPase from Neurospora crassa, the two most prominent components are polypeptides of Mr = 70,000 and 60,000. We previously reported the isolation of the gene vma-1, which encodes the Mr = 70,000 polypeptide, and presented evidence that the polypeptide contains the site of ATP hydrolysis (Bowman, E. J., Tenney, K., and Bowman, B. J. (1988) J. Biol. Chem. 263, 13994-14001). We now report the isolation of a gene (designated vma-2), that encodes the Mr = 60,000 polypeptide. Analysis of the DNA sequence shows that the polypeptide has 513 amino acids and a molecular mass of 56,808 daltons (and will thus be referred to as the 57-kDa polypeptide). It is fairly rich in polar amino acids and has no apparent membrane-spanning domains. The vma-2 gene contains five short introns (55-71 bases), all clustered in the 5' end of the coding region. The gene maps to the right arm of linkage group II, near 5 S RNA gene 3. Thus, it is unlinked to vma...

Fragments, generated by restriction enzyme digestion, of the 58-kilobase Enterococcus (Streptococcus) faecalis tetracycline resistance plasmid pCF10 were cloned and introduced into Escherichia coli and E. faecalis to characterize the pheromone-inducible conjugation system encoded by this plasmid. Western blot (immunoblot) analyses revealed that a 130-kilodalton (kDa) antigen, identical to the Tra130 antigen shown previously to be involved in pCF10-mediated pheromone-inducible surface exclusion, was produced by both bacterial hosts carrying the recombinant plasmid pINY1825 (cloned EcoRI C fragment). Both bacterial hosts carrying pINY1825 also produced various amounts of immunologically related 118- to 125-kDa antigens (designated pre-Tra130) that resembled antigens produced by E. faecalis cells carrying pCF10. An additional 150-kDa antigen, Tra150, probably involved in pheromone-induced cellular aggregation, was produced by Escherichia coli and E. faecalis hosts carrying pINY1801 (cl...

Mouse cells are nonpermissive for simian virus 40 (SV40); replication of viral DNA is undetectable and progeny virions are not produced. Infection leads instead to the establishment of stably transformed cell lines in which viral DNA is covalently integrated into cellular DNA. We have followed the fate of SV40 DNA in infected mouse cells to define steps in viral DNA metabolism that precede integration. A novel high molecular weight form of SV40 DNA is synthesized shortly after infection by a process sensitive to the inhibition of DNA replication. This DNA represents polymers in which viral genomes are organized as tandem "head-to-tail" arrays. Recombination can be demonstrated with mutant viruses, but the recombination frequency is not high enough to account for the synthesis of polymers by recombination between infecting genomes. We conclude that polymers are synthesized by DNA replication and that they then recombine with one another. We believe that the polymers also re...

A series of nucleocapsid protein (NP)-deleted genes of the Onderstepoort strain was constructed in order to locate antigenic regions of the NP of canine distemper virus. The expression of proteins from 5 H-deleted NP genes was examined in COS-7 cells by indirect immunofluorescence assay using three monoclonal antibodies (MAbs), c-5, f-5 and h-6, and a rabbit serum against NP. These MAbs reacted with two regions of NP. Amino acid residues from 1 to 80, and 337±358, were necessary and sufficient for formation of the epitopes identified by MAbs f-5 and h-6, and c-5, respectively. The proteins translated from intact or 3 H-deleted genes were found to be localized in the nuclei of COS-7 cells, whereas the proteins from the 5 H-deleted genes were mainly detected in the cytoplasm. These results suggested that 80 amino acid residues at the N-terminus are required for transportation of NP into the nucleus.

The haemolytic plasmid pSU316 is incompatible with members of the IncFIII and IncFIV incompatibility groups. Plasmid pSU307 (pSU316 hlyC : : Tn5) was inserted by integrative suppression into the chromosome of JW 112, a temperature-sensitive dnaA mutant of Escherichia coli. The incompatibility properties of this strain (SU5 1) were studied and it was found that : (1) plasmid pSU306 (pSU316 hlyA : : Tn802) was rapidly lost from strain SU51 both at 30 "C and 42 "C; (2) the IncFIII plasmid pSU397 (ColB-K98 : :Tn802) was lost from strain SU51 at 42 "C but not at 30 "C; and (3) the IncFIV plasmid R124 was stably maintained in strain SU51 at both temperatures. Revertants of pSU307 to the autonomous state could be obtained from SU5 1. These revertants exerted incompatibility towards the prototype plasmids pSU306, pSU397 and R124 in the same way as pSU307 itself. Thus, strain SU51 provided a suitable method for distinguishing the three different incompatibility determinants of plasmid pSU3 16.

The mechanisms of chloroplast recombination are largely unknown. Using the chloroplast-encoded homing endonuclease I-CreI from Chlamydomonas reinhardtii, an experimental system is described that allows the study of double strand break (DSB)-induced recombination in chloroplasts. The I-CreI endonuclease is encoded by the chloroplast ribosomal group I intron of C.reinhardtii and cleaves specifically intronless copies of the large ribosomal RNA (23S) gene. To study DSB-induced recombination in chloroplast DNA, the genes encoding the I-CreI endonuclease were deleted and a target site for I-CreI, embedded in a cDNA of the 23S gene, was integrated at an ectopic location. Endonuclease function was transiently provided by mating the strains containing the recombination substrate to a wild-type strain. The outcome of DSB repair was analyzed in haploid progeny of these crosses. Interestingly, resolution of DSB repair strictly depended upon the relative orientation of the ectopic ribosomal cDNA and the adjacent copy of the 23S gene. Gene conversion was observed when the 23S cDNA and the neighbouring copy of the 23S gene were in opposite orientation, leading to mobilization of the intron to the 23S cDNA. In contrast, arrangement of the 23S cDNA in direct repeat orientation relative to the proximal 23S gene resulted in a deletion between the 23S cDNA and the 23S gene. These results demonstrate that C.reinhardtii chloroplasts have an efficient system for DSB repair and that homologous recombination is strongly stimulated by DSBs in chloroplast DNA.

The transition from A to G at nt 5656 5656A™ G in mitochondrial DNA has been suggested to be a pathogenic mutation and, furthermore, a heteroplasmic one. We found that the mutation was present in 14 out of 83 healthy controls from northern Finland and that 5656A™ G was exclusively associated with mtDNA haplogroup U. Interestingly, 5656A™ G appeared to be heteroplasmic in NheI digestion of PCR fragments that were amplified by using a mismatched oligonucleotide primer creating a digestion site in the presence of the mutant variant. However, we did not detect the wild type genome in clones from such a sample and subsequent experiments revealed that the apparent heteroplasmy was due to inhibition of NheI by NaCl. Our results suggest that 5656A™ G is a polymorphism and it may be highly characteristic for Finns. Furthermore, new heteroplasmic mutations identified by restriction fragment analysis should be adequately controlled for any false positive results that may be due to incomplete digestion.

Whole genome shotgun sequence analysis has become the standard method for beginning to determine a genome sequence. The preparation of the shotgun sequence clones is, in fact, a biological experiment. It determines which segments of the genome can be cloned into Escherichia coli and which cannot. By analyzing the complete set of sequences from such an experiment, it is possible to identify genes lethal to E. coli. Among this set are genes encoding restriction enzymes which, when active in E. coli, lead to cell death by cleaving the E. coli genome at the restriction enzyme recognition sites. By analyzing shotgun sequence data sets we show that this is a reliable method to detect active restriction enzyme genes in newly sequenced genomes, thereby facilitating functional annotation. Active restriction enzyme genes have been identified, and their activity demonstrated biochemically, in the sequenced genomes of Methanocaldococcus jannaschii, Bacillus cereus ATCC 10987 and Methylococcus capsulatus.

Since the discovery of plasmid, various methods have been developed to isolate plasmid DNA. All the methods have one common and important target of isolating plasmid DNA of high quality and quantity in less time. These methods are not completely safe because of use of toxic chemicals compounds. The developed protocol for plasmid extraction is based on the alkaline lysis method of plasmid preparation (extraction atpH 8.0) with slight modifications. Cell lysis reagent sodium dodecyl sulfate is replaced by lipase enzyme present in laundry detergent. A good plasmid preparation can be made, which is well suited for subsequent molecular biology applications. By taking safety measures on count, contaminants like, RNA and protein can be completely avoided with maximized plasmid yield. The resultant plasmid quality and quantity can be well comparable to other prevalent methods.

The recombinant plasmid pAG0 contains the Herpes simplex virus type 1 (HSV-1) thymidine kinase (TK) gene and consists of a 2-kb PvuII fragment of HSV-1 DNA inserted into the PvuII site of pBR322. A deletion mutant of PAGO, designated pMHl10, has been isolated which removes the normal HSV-1 TK gene promoter but places the promoter of the pBR322 tetracycline-resistance (tet*) gene only about 400 bp from the translational start codon of the HSV-I TK polypeptide. In contrast to PAGO, which transforms mouse LM(TK-) cells to TK' but is only weakly expressed in TK-bacteria, pMHll0 not only efficiently transforms LM(TK-) cells to TK' but also enables TK-Escherichia coli K-12 cells to form colonies on selective plates containing S-fluorodeoxyuridine (FdUrd) plus thymidine (dThd) and to exhibit fully restored ability to incorporate [3H] dThd into DNA. The levels of TK activity expressed by bacteria harboring pMHl10 were about as high as those expressed by bacteria harboring plasmid pTK3, which contains the wild-type E. coli TK gene. The TK activity expressed in bacteria harboring pMHll0 was partially purified and shown to be HSV-1-specific by serological and disc PAGE analyses and by experiments demonstrating that this enzyme phosphorylated ['*'I]deoxycytidine.