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Sensitive methods for detecting coliform bacteria, and pathogenic bacteria (Salmonella and Shiqella) in environmental waters that do not require culturing of bacteria were developed by using the polymerase chain reaction (PCR) and gene probes. Cells were collected by filtration and DNA was released by freeze-thaw cycling. PCR amplification of region of lacZ gene was used as a target for detection of total coliform bacteria. A region of the lamB gene was the target for detection of Escherichiacoli, Salmonella and Shigella. A region of the uid gene was used for detection of E. coli and Shigella. Biotin was incorporated during PCR amplification and the biotinylated DNA was detected by using membrane-bound poly-T-tailed capture probes. As little as 1 fg of total DNA and as few as 1 cell per 100 ml were detectable by the PCR-gene probe method. [ABSTRACT FROM AUTHOR]

For effective control of the vector-borne bovine diseases, a thorough knowledge of the nature of the infective agents, the vectorial capability, and the susceptibility of the bovine host is required. There is, however, little information on prevalence and transmission of bovine diseases due to the difficulty of identifying infected animals by conventional microscopic examination. Therefore, we have used the novel technology of diagnostic DNA-probes for detection of Babesia infection. The best probe can detect as little as 25 ng of purified Babesia DNA, the equivalent of 0.001% parasitemia. Each of the developed probes are species specific. Research on development of vaccines against other tick-borne diseases is also being pursued. The strategy is to identify surface proteins relevant to protection, clone them, and express gene coding for these proteins and test their effectiveness as protective immunogens in recombinant vaccinia construct. [ABSTRACT FROM AUTHOR]

It is now over 30 years since the cholera enterotoxin was discovered in India and more than 20 years since it was first purified to homogeneity. Early expectations that, as with diphtheria and tetanus, a toxoid vaccine would solve the cholera problem have not been fulfilled. We are examining the reasons why cholera toxin (CT) vaccines have failed to provide effective immunity. Studies with polyclonal (pAbs) and monoclonal (mAbs) antibodies reveal significant differences among the CTs and CT-related enterotoxins (LTs). All field trials of toxoid vaccines have used CT-1 antigen against the prevailing epitype of CT, usually CT-2. Single amino acid substitutions in the immunodominant B-subunit protein have profound effects on immunological reactivity. We have employed pAbs and mAbs, genetically engineered chimeric B-subunit proteins, checkerboard immunoblotting (CBIB), synthetic peptides and their antisera, and sequential overlapping synthetic hexapeptides representing the B-subunit chain to identify epitopes in the CT family. Reactivity of some, but not all, synthetic hexapeptides with epitope activity with polyclonal antisera can be reduced by pretreatment of the serum with the native toxin protein indicating that certain continuous epitopes are exposed on the surface of the native protein. Interestingly, the holotoxin was more effective in this regard than the native B-subunit pentamer indicating a conformational difference between the two. Human convalescent sera gave diffuse patterns of reactivity with discontinuous (conformational) epitopes. A major tetrapeptide epitope has been identified in a conserved region of the protein. Substitution of amino acids in this region can be permissive or can reduce or eliminate activity. Studies like these may lead to the development of rational synthetic peptide vaccines, but a vaccine composed only of CT antigen can only protect against severe life-threatening diarrhea, not all the diarrhea caused by cholera vibrios. [ABSTRACT FROM AUTHOR]

A multicopy phenotype approach to identify new regulatory proteins is not without drawbacks. Overexpression of an activity is not a "real life" situation for the cell. Hartwell and coworkers remark that this method, even while conveniently providing the cloned wildtype gene for further analysis, might fool the investigator; overexpression of any activity might give the desired phenotype, or lead to cell death (2). However, there are ways to circumvent these problems and the investigator need only realize that, when using a multicopy phenotype approach, the next obvious step after the identification of an activity will be to determine its role, as a single copy function, in the cell. [ABSTRACT FROM AUTHOR]

Extraction of S-layer protein by treatment with a 6M urea indicated that there appeared to be extra-high molecular weight protein in the extracts obtained from Bacillus thuringiensis subsp. israelensis (B.t.i.) strain 4Q2. The antibody toward this large molecular weight protein was prepared and used for locating of S-layer protein on B.t.i. by using indirect immunofluorescent technique. Immunodiffusion reaction and Western blot analysis confirmed the specificity of the anti-S-layer protein. It was found that the antibody against S-layer protein inhibited the plasmid transfer via the conjugation-like process. The frequency of transfer of plasmid pBC16 was found to be 9.7×10-6 and less than 10-8 in the presence and absence of anti-S-layer antibody, respectively. Using antibody detection technique, S-layer protein gene from B.t.i. strain 4Q2 was cloned in phagemid pKSII and plasmid pUC12 of E.coli DH5α. Three positive clones containing the gene encoding for the S-layer protein were obtained. Two clones had the insert of 5.2 kb, and the other one had an insert of 6.6 kb. The expression of the S-layer protein gene was detected by using Western blot analysis. [ABSTRACT FROM AUTHOR]

The role of crb-86 in cat-86 induction may be a function of both its complementarity with 16S rRNA and the crb-@#@ encoded peptide. Solving the role of crb. as a gene regulatory device will dissect ribosome function in a manner not possible by conventional methodology much in the same vein as other recently discovered variations of ribosome function (12). [ABSTRACT FROM AUTHOR]

The increased availability and a variety of recombinant DNA techniques, has provided a route for the genetic analysis of newly isolated bacteria. The data so obtained is essential if the manipulation of metabolic pathways of these strains is to be successful. Three general conclusions are possible. Using genetically well characterized models, effective procedures for the genetic analysis of newly isolated bacteria can be developed. Next, a combination of physical and genetic techniques provides genetic analysis superior to the more traditional procedures. Finally, improved techniques enables whole genome analysis to be applied to a range of organisms. The unveiling of hidden complexities in the manner in which bacteria store their biological information can only contribute to our ability to manipulate them for practical purposes. [ABSTRACT FROM AUTHOR]

Exposure of Escherichia coli to agents that damage DNA or interfere with DNA replication results in the induction of the SOS response. A number of chromosomal genes that are repressed by the LexA protein are transcribed at higher levels and various lysogenic bacteriophage are induced. The RecA protein becomes activated by binding to some intracellular inducing signal, probably single-stranded DNA and then mediates proteolytic cleavage of LexA and bacteriophage repressors by facilitating an otherwise latent capacity of these molecules to autodigest. The products of the SOS-regulated operon umuDC axe required for most UV and chemical mutagenesis. We have shown that the UmuD protein shares homology with the carboxyl-terminal domains of LexA and several bacteriophage repressors and is activated for its role in mutagenesis by a RecA-mediated proteolytic event. Thus the regulation of umuD involves a transcriptional derepression and a posttranslational activation that are mechanistically and evolutionary related. A set of missense mutants of umuD was isolated and shown to encode mutant UmuD proteins that are deficient in RecA-mediated cleavage in vivo but which can be partially cleaved at a higher UV dose. Most of these mutations are dominant to umuD* with respect to UV mutagenesis yet do not interfere with SOS induction. Although both UmuD and UmuD' form homodimers, we have found evidence that they preferentially form heterodimers. These studies of umuD have suggested a role for intact UmuD in the modulation of SOS mutagenesis. Other genetic studies have indicated that the RecA protein plays a third role in mutagenesis besides mediating the cleavage of LexA and UmuD. In addition, we have observed that efficiency of UV mutagenesis is greatly reduced by mutations affecting the groESand groEL heat-shockgenes. These genes encode proteins that function as molecular chaperones which mediate protein folding and protein-protein interaction. It seems possible that they may play a role in the proper assembly of a protein complex required for SOS mutagenesis. [ABSTRACT FROM AUTHOR]

The lignin-degrading enzyme system of white-rot fungi, which are mostly basidiomycetes, has been studied intensively in recent years. The extracellular component of the system is comprised of lignin peroxidase, manganese peroxidase, glyoxal oxidase and certain metabolites. Lignin is fragmented by this system, and the plethora of degradation products taken up by the hyphae and further metabolized by the intracellular system. The intracellular system has received very little research attention. The structural complexity and heterogeneity of lignin show in fact that this enzyme system is so nonspecific that it also degrades a variety of hazardous compounds, including polycyclic aromatics, some polychlorinated biphenyls and dioxins, DDT, and many chlorinated phenols. Using pentachlorophenol (PCP) as a model substrate, we have studied the possibility of using white-rot fungi to remediate soils contaminated with hazardous compounds. Successful laboratory results led to a field study in the summer of 1989. Results showed that the laboratory findings could be duplicated in the field. [ABSTRACT FROM AUTHOR]

Bacterial plasmids contain genetic determinants for resistance systems for Hg2+ (and organomercurials), Cd2+, AsO2, AsO43-, CrO42-, TeO32-, Cu2+, Ag+, Co2+, Pb2+, and other metals of environmental concern. In some cases, there is the potential for using genetically engineered microbes for bio-remediation. Recombinant DNA analysis has been applied to mercury, cadmium, zinc, cobalt, arsenic, chromate, tellurium and copper resistance systems. The eight mercury resistance systems that have been sequenced all contain the gene for mercuric reductase, the enzyme that converts toxic Hg2+ ions to less toxic volatile metallic Hg°. Four of these systems also determine the enzyme organomercurial lyase, which cuts the HgC bond and thus detoxifies methylmercury and phenylmercury. Two sequenced Cd2+ resistance determinants govern cellular efflux of Cd2+ assuring a low level of intracellular Cd2+: not an obvious candidate for bioremediation. Cadmium accumulation by bacterial metallothionein or phytochelatin is a potentially useful process, but only preliminary reports have appeared on bacteria producing polythiol polypeptides. For arsenic resistance, a unique efflux ATPase maintains low intracellular As levels. A bacterial AsO2- oxidase has been reported, with the potential of converting more toxic As(III) into less toxic As(V), but this system has not been studied in recent years. For chromate, resistance results from reduced cellular uptake. However, both soluble and membrane-bound Cr(VI) reductase bacterial activities convert more toxic Cr(VI) to less toxic Cr(III) in different bacteria. [ABSTRACT FROM AUTHOR]

DNA probes are useful markers for studying pathogen population genetics. Repetitive probes may produce characteristic "fingerprints" that identify strains or lineages of asexually-reproducing pathogens. Relatedness among strains can be inferred, giving insight into the processes of race evolution, gene flow, and other aspects of pathogen population biology. Information about pathogen population dynamics may be useful in designing improved strategies for disease management. Knowledge of pathogen lineages can also be used in the selection of diverse tester strains used in screening of breeding material for crop improvement. Different probes may reveal somewhat different information about a population. The specific estimates of diversity obtained for a given population depend on the specific probe employed in the analysis. Probes may vary in the extent to which they reveal RFLP patterns that are associated with pathogenic race. If there is a perfect association between RFLP type and race, a probe may be used to identify race. The extent of association between identified molecular and pathogenic phenotypes depends upon the characteristics of the population tested, of the differential cultivars chosen for virulence analysis, and of the probe and enzyme used for the analysis of RFLP type. [ABSTRACT FROM AUTHOR]

The survival of microorganisms is dependent on their ability to respond to a changing environment. In the very stressed environment of the CF lung, with salty and dehydrated mucus, the microorganisms need to protect themselves from losing their intracellular water and one way to accomplishing this is to produce an exopolysaccharide capsule with strong gelling properties as a barrier to dehydration. It is interesting that the algD promoter is activated by those environmental factors that are characteristic of the CF disease, which explains why CF patients are particularly vulnerable to infections by mucoid P. aeruginosa. It is also interesting to note that the alginate capsule, which is presumably produced to protect P. aeruginosa from intracellular dehydration, also affords protection The activation of the algD promoter has been measured by measuring catechol 2,3-dioxygenase (C230) activity, since a construct containing the xylE gene placed under the algD promoter was used in these experiments (see ref. 18 and 19). One unit of catechol 2,3-dioxygenase activity is defined as the amount of enzyme oxidizing one mmol of catechol to 2-hydroxymuconic semialdehyde, a product with a molar extinction coefficient of 4.4 × 104 at 375 nm. against antibiotics and antibodies to the detriment of the human patients. When the environment is beset with chlorinated compounds, the immediate response of natural microorganisms is to evolve degradative genes in the form of a plasmid. As a first step, they tend to recruit genes that allow degradation of a structurally analogous non-chlorinated compound, which undergo mutational or recombinational divergence to the appropriate genes whose products have broad substrate specificities to include chlorinated compounds. Such evolutionary processes apparently are not very effective for highly chlorinated compounds for which appropriate enzyme systems have not yet fully developed. In a single incidence of directed evolution, the chromosomal DNA shows the presence of multiple copies of a transposable element near the evolved genes, suggesting that an accelerated process of evolution may bypass the requirement of genetic relatedness of the evolved genes to the genome of the recruiting cells. [ABSTRACT FROM AUTHOR]

Lectins are multivalent carbohydrate-binding proteins and glycoproteins. In rice and other cereal crops, lectins are well characterized for their binding specificity to N-acetylglucosamine or its oligomers. Although the natural function of lectin remains unknown, several possible functions of cereal lectins have been proposed including an involvement in plantmicrobial interaction. We found that rice lectins are not exclusively confined to embryo, but also in seedling leaf and root, especially at the root tip, and opening stoma of young leaf, which are preferent adhesion sites of microbes. Lectin contents varied with developmental stage and environmental factors. Different rice varieties contain different amount of lectin and respond differently to inoculation by associative nitrogen-fixing Klebsiella R15 and R17. Better plant growth promotion and associative nitrogen-fixation were observed in high lectin variety. Colonization of bacteria on the root surface increased the root lectin. Our results support for the potential application of nitrogen-fixing Klebsiella for promotion of growth and associative nitrogen fixation in rice, Oryza sativa L. [ABSTRACT FROM AUTHOR]

The formation of hyperplasias termed "galls" produced by P.savastanoi and the formation of crown gall tumors by Agrobacterium tumefaciens have a number of features in common, In both cases: the unregulated production of IAA arid cytokintn at the site of-infection is-required for gaii formation. Thus, both compounds, the product of bacteria! genes, are virulence factors in both diseases, and therefore, it is not surprising that the disease symptoms are similar. Further, both violence factors are codecs by genes on large plasmids and have a remarkable degree of homology with each other. However, in Pseudcmonas the genes are constitutively expressed in the bacterium, whereas in Agrobacterium. expression occurs oniy after the genes are integrated into the plant chromosome. Apparently, the structura, component of the gene is prokaryotic in nature whereas the upstream regulatory sequences are eukaryotic and respond to plant transcription and translation machinery. Disease production:by P. savssianos appears to be a much simpler process than that by Agrohacterium. in the former ease, the two phytohormones are synthesized by the bacteria at the site of infection and flying bacteria are necessary for continued symptomatology. On the other hand, Agrobactenaum has evolved a very compfex mechanism.for introducing a piece of its plasmid into plant ceiis where it functions. Consequently, the continuecl growth of the bacteria at the wound site is not necessary for tumor formation. The vir genes of Agrobacte rium apparently do not have a counterpart in .Pseudomonas. although its virulence piasmid has not been subjected to extensive mutagenesis. [ABSTRACT FROM AUTHOR]

We have isolated 5 genomic DNAs (prxC1a. prxC1b. prxC1c. prxC2 and prxC3 @#@) coding for horseradish peroxidase (HRP). All 5 genes consisted of 4 exons and 3 introns, and the positions of introns in the coding regions were same in all genes. Putative promoter sequences and polyadenylation signals were also found. Two peroxidase genes (prxCa and prxEa) of Arabidopsis thaliana which belongs to the same family of horseradish, Brassica.were also cloned. The structures and sequences of the coding regions of prxCa. and prxEa were very similar to prxC1b and prxC3, respectively. Transcripts of all HRP genes were abundant in horseradish cultured cells, but organspecific expression was observed in horseradish plant. prxC1 was expressed in root and stem, C2 in stem, and C3 in root. 5'-Noncoding regions of HRP isozyme genes were joined to β-glucuronidase (GUS) reporter gene and introduced into tobacco protoplast by electroporation. Promoter activity was detected in every construct, especially prxC2 gene showed several times higher activity of CaMV35S promoter. The promoter-GUS fused genes were transferred to tobacco leaf disk using binary vector system with Aqrobacteriumtumefaciens. Strong GUS expression was observed in root of transgenic tobacco, but not in leaf. When callus was induced from leaf, GUS activity enhanced 15 -50 time of that in leaf in all promoter regions as observed in horseradish callus. [ABSTRACT FROM AUTHOR]

With the development of techniques for the introduction of foreign genes into plant cells and the regeneration of transgenic plants, a number of different approaches have been taken to attempt to produce resistance to virus diseases. Those most commonly used have been (1) the expression of antisense RNAs; (2) the expression of satellite RNAs and defective interfering RNAs and DNAs; and (3) the expression of genes encoding viral capsid (coat) proteins. Of these the latter has been generally accepted because of its ready application to control a variety of virus diseases in a number of different crops. Because we recently published a review of the current state of the field (Beachy et al, 1990) this brief review will highlight the status of the field. [ABSTRACT FROM AUTHOR]

The yeast cell wall is composed mainly of glucan, mannoprotein and chitin (1). In most yeasts, the polysaccharide glucan is predominantly β-1,3-linked with some branching via (3-1,6- linkages (2). The glucan network in the yeast cell wall, due to its rigidity, is an essential structure in preventing lysis of the protoplast in a hypotonic environment. Several microorganisms have been reported to produce extracellular enzymes capable of lysing yeast cells (3,4). Analysis of the constituents of these lytic enzyme preparations revealed the presence, among other activities, of β-1,3-glucanases (3,5). When combinedwith athiol reagent, β-1,3-glucanase alone was found to be able to lyse the yeast cell (6). Currently, the most common-used enzyme for digestion of yeast cell walls is an enzyme preparation called Zymolyase, produced from Oerskovia xanthineolytica (5). We have isolated the glucanase gene from this organism and completely determined its sequence (7). [ABSTRACT FROM AUTHOR]

The coding sequence of bifunctional thymidylate synthase-dihydrofolate reductase (TS-DHFR) from moderate pyrimethamine resistant strain of Plasmodium falciparum was assembled in a pUC expression vector. Insertion of a synthetic oligonucleotide duplex containing a ribosomal binding sequence at 5' upstream to ATG start codon resulted in a recombinant plasmid which expressed catalytically active TS-DHFR in E. coli. The expression of Plasmodium enzyme was demonstrated as follows: First, transformation of a TS-deficient E. coli resulted in genetic complementation of the deficiency and the activities of TS and DHFR were significantly higher than those in control host extracts. Second, TS and DHFR activities copurified upon chromatography with an apparent molecular weight ∼ 110 kDa, a characteristic of protozoan bifunctional enzyme. Third, the enzyme formed a covalent complex with [3H] FdUMP-CH2H4folate, and SDS polyacrylamide gel electrophoresis showed the subunit of the recombinant enzyme to be 67 kDa. Finally, N-terminus sequencing of the 67 kDa protein indicated that the N-terminus was not blocked and the sequences of the first 10 amino acids perfectly matched the reported nucleotide sequence. The recombinant enzyme was purified to homogeneity using 10 formylfolate affinity chromatography followed by FPLC on Mono Q column. Since the coding sequence possesses unique Nco1 and Xba1 sites which flank 243 bp of the DHFR gene that include point mutations thus far linked to pyrimethamine resistance, recombinant plasmids of wild type (3D7) and highly pyrimethamine resistant (7G8) gene were constructed by cassette mutagenesis using Nco1 -Xba1 fragments from the corresponding cloned TS-DHFR genes. Characterization of the purified-recombinant enzymes with regard to their kinetic properties and inhibition by pyrimethamine strongly clearly indicated that point mutations are the molecular basis of pyrimethamine resistance in P. falciparum. [ABSTRACT FROM AUTHOR]

A heterogeneous group of crystallins comprise 80-90% of the soluble protein of the ocular lens and are responsible for lens transparency (see 30 for review). The two a-crystallins (αA and (αB) are present in all vertebrates, and arose from duplication of a common ancestral gene. The αA gene is expressed in a lens-specific manner, while the αB gene is expressed in numerous tissues (see 24 for review). A combination of transgenic mouse (28), mutagenesis, and cDNA cloning experiments (20) have implicated the αA-CRYBP1 site (5′- GGGAAATCCC-3′ at-66 to-57) in the lens-specific expression of the mouse α-crystallin gene. [ABSTRACT FROM AUTHOR]

In Candida maltosa, expression of many genes is induced by the hydrophobic compound, n-alkane, resulting in biogenesis of organelles such as endoplasmic reticulum and peroxisome where n-alkane is metabolized. Host-vector systems are needed in order to investigate this phenomenon at the molecular level. Since C. maltosa does not have plasmids, the isolation of an ARS site from the genome of C maltosa was attempted so as to construct vectors. Several host-vector systems were constructed to promote reverse genetics in this non-conventional yeast as described in our recent papers shown below. [ABSTRACT FROM AUTHOR]

The first moveable DNA elements to be studied at the molecular level were the transposable elements found in prokaryotes. Although several different types are now known, they all share two properties: first, the DNA within the element encodes a gene or genes that are required for transposition, and, second, specific DNA sequences are repeated in inverted orientation at the two ends of the element and are required for transposition. [ABSTRACT FROM AUTHOR]

Biochemistry and genetics epitomize what is called the reductionist approach to biology; single biological elements (for example, a protein, an enzyme, or a gene) are examined in great detail through a series of narrowly defined questions and experimental protocols. This approach can illuminate many phenotypic characteristics of complex systems, including whole organisms, if those phenotypes are the direct consequence of a single gene's function. [ABSTRACT FROM AUTHOR]