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Aristeromycin is a unique carbocyclic nucleoside antibiotic produced by Streptomyces citricolor. In order to elucidate its intriguing carbocyclic formation, we used a genome-mining approach to identify the responsible enzyme. In silico screening with known cyclitol synthases involved in primary metabolism, such as myo-inositol-1-phosphate synthase (MIPS) and dehydroqunate synthase (DHQS), identified a unique MIPS orthologue (Ari2) encoded in the genome of S. citricolor. Heterologous expression of the gene cluster containing ari2 with a cosmid vector in Streptomyces albus resulted in the production of aristeromycin, thus indicating that the cloned DNA region (37.5 kb) with 33 open reading frames contains its biosynthetic gene cluster. We verified that Ari2 catalyzes the formation of a novel five-membered cyclitol phosphate from d-fructose 6-phosphate (F6P) with NAD + as a cofactor. This provides insight into cyclitol phosphate synthase as a member of the MIPS family of enzymes. A biosynthetic pathway to aristeromycin is proposed based on bioinformatics analysis of the gene cluster., (© 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

As baculoviruses usually have a narrow insecticidal spectrum, knowing the mechanisms by which they control the host-range is prerequisite for improvement of their applications as pesticides. In this study, from supernatant of culture cells transfected with DNAs of an Autographa californica multiple nucleopolyhedrovirus (AcMNPV) mutant lacking the antiapoptotic gene p35 (vAc(∆P35)) and a cosmid representing a fragment of Spodoptera exigua nucleopolyhedrovirus (SeMNPV), a viral strain was plaque-purified and named vAcRev. vAcRev had a broader host range than either vAc(∆P35) or SeMNPV parental virus, being able to infect not only the permissive hosts of its parental viruses but also a nonpermissive host (Spodoptera litura). Genome sequencing indicated that vAcRev comprises a mixture of two viruses with different circular dsDNA genomes. One virus contains a genome similar to vAc(∆P35), while in the other viral genome, a 24.4 kbp-fragment containing 10 essential genesis replaced with a 4 kbp-fragment containing three SeMNPV genes including a truncated Se-iap3 gene. RNA interference and ectopic expression assays found that Se-iap3 is responsible for the host range expansion of vAcRev, suggesting that Se-iap3 inhibits the progression of apoptosis initiated by viral infection and promotes viral propagation in hosts both permissive and non-permissive for AcMNPV and SeMNPV.

In the course of our search for anti-dormant Mycobacterial substances, nybomycin (1) was re-discovered from the culture broth of a marine-derived Streptomyces sp. on the bioassay-guided separation. Compound 1 showed anti-microbial activity against Mycobacterium smegmatis and Mycobacterium bovis BCG with the MIC of 1.0μg/mL under both actively growing aerobic conditions and dormancy inducing hypoxic conditions. Compound 1 is also effective to Mycobacterium tuberculosis including the clinically isolated strains. The mechanistic analysis indicated that 1 bound to DNA and induces a unique morphological change to mycobacterial bacilli leading the bacterial cell death., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

During the past few decades, numerous plasmid vectors have been developed for cloning, gene expression analysis, and genetic engineering. Cloning procedures typically rely on PCR amplification, DNA fragment restriction digestion, recovery, and ligation, but increasingly, procedures are being developed to assemble large synthetic DNAs. In this study, we developed a new gene delivery system using the integrase activity of an integrative and conjugative element (ICE). The advantage of the integrase-based delivery is that it can stably introduce a large DNA fragment (at least 75 kb) into one or more specific sites (the gene for glycine-accepting tRNA) on a target chromosome. Integrase recombination activity in Escherichia coli is kept low by using a synthetic hybrid promoter, which, however, is unleashed in the final target host, forcing the integration of the construct. Upon integration, the system is again silenced. Two variants with different genetic features were produced, one in the form of a cloning vector in E. coli and the other as a mini-transposable element by which large DNA constructs assembled in E. coli can be tagged with the integrase gene. We confirmed that the system could successfully introduce cosmid and bacterial artificial chromosome (BAC) DNAs from E. coli into the chromosome of Pseudomonas putida in a site-specific manner. The integrase delivery system works in concert with existing vector systems and could thus be a powerful tool for synthetic constructions of new metabolic pathways in a variety of host bacteria.

In previous work transduction of Escherichia coli with phage λ particles carrying packaged plasmids was shown to provide complete off-to-on expression of plasmid-borne genes (Cronan, J.E., 2003. J. Bacteriol. 185, 6522-6529). The plasmids used contained the phage λcos site (and hence are cosmids) and were very efficiently packaged into λ phage particles in vivo upon induction of λ lysogens having an inactivated cos site. However, a shortcoming was that the stocks contained a variable fraction of infectious λ phage which arose by recombinational repair of the inactive cos site. I report that the construction of E. coli strains that eliminate the background of infectious phage and show that the system can be utilized to express proteins by the phage T7 RNA polymerase/pET vector system., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Only a small fraction of the bacterial diversity present in natural microbial communities is regularly cultured in the laboratory. Those bacteria that remain recalcitrant to culturing cannot be examined for the production of bioactive secondary metabolites using standard pure-culture approaches. The screening of genomic DNA libraries containing DNA isolated directly from environmental samples (environmental DNA (eDNA)) provides an alternative approach for studying the biosynthetic capacities of these organisms. One drawback of this approach has been that most eDNA isolation procedures do not permit the cloning of DNA fragments of sufficient length to capture large natural product biosynthetic gene clusters in their entirety. Although the construction of eDNA libraries with inserts big enough to capture biosynthetic gene clusters larger than ∼40kb remains challenging, it is possible to access large gene clusters by reassembling them from sets of smaller overlapping fragments using transformation-associated recombination in Saccharomyces cerevisiae. Here, we outline a method for the reassembly of large biosynthetic gene clusters from captured sets of overlapping soil eDNA cosmid clones. Natural product biosynthetic gene clusters reassembled using this approach can then be used directly for functional heterologous expression studies., (Copyright © 2012 Elsevier Inc. All rights reserved.)

The expression of a gene or a set of genes from one organism in a different species is known as "heterologous expression." In actinomycetes, prolific producers of natural products, heterologous gene expression has been used to confirm the clustering of secondary metabolite biosynthetic genes, to analyze natural product biosynthesis, to produce variants of natural products by genetic engineering, and to discover new compounds by screening genomic libraries. Recent advances in DNA sequencing have enabled the rapid and affordable sequencing of actinomycete genomes and revealed a large number of secondary metabolite gene clusters with no known products. Heterologous expression of these cryptic gene clusters combined with comparative metabolic profiling provides an important means to identify potentially novel compounds. In this chapter, the methods and strategies used to heterologously express actinomycete gene clusters, including the techniques used for cloning secondary metabolite gene clusters, the Streptomyces hosts used for their expression, and the techniques employed to analyze their products by metabolic profiling, are described., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Two endogenous plasmids are present in Synechococcus elongatus PCC 7942, a model organism for studying photosynthesis and circadian rhythms in cyanobacteria. The large plasmid, pANL, was shown previously to be involved in adaptation of S. elongatus cells to sulfur starvation, which provided the first evidence of cellular function of a cyanobacterial plasmid. Here, we report the complete sequence of pANL, which is 46,366 bp in length with 53% GC content and encodes 58 putative ORFs. The pANL plasmid can be divided into four structural and functional regions: the replication origin region, a signal transduction region, a plasmid maintenance region, and a sulfur-regulated region. Cosmid-based deletion analysis suggested that the plasmid maintenance and replication origin regions are required for persistence of pANL in the cells. Transposon-mediated mutagenesis and complementation-based pANL segregation assays confirmed that two predicted toxin-antitoxin cassettes encoded in the plasmid maintenance region, belonging to PemK and VapC families, respectively, are necessary for plasmid exclusion. The compact and efficient organization of sulfur-related genes on pANL may provide selective advantages in environments with limited sulfur.

Rhodococcus opacus strain SAO101 was shown to degrade on various polycyclic aromatic hydrocarbons such as naphthalene, dibenzofuran (DF), and dibenzo-p-dioxin (DD). One of the unique traits of the strain SAO101 is its ability to oxidize DF compounds by lateral dioxygenation. To clone the lateral dioxygenase gene involved in compound degradation in strain SAO101, we identified a cosmid clone that oxidizes aromatic compounds by using SAO101 genomic DNA. Sequencing analysis revealed that isolated cosmid clone contained ring-hydroxylating dioxygenase genes (narAaAb) with homologies to indene dioxygenase genes of Rhodococcus strain I24 and naphthalene dioxygenase genes of Rhodococcus strain NCIMB12038. The NarAaAb-expressing Rhodococcus cells exhibited broad substrate specificity for bicyclic aromatic compounds and had high ability to degrade dibenzofuran and naphthalene. Metabolite analysis revealed that dihydrodiol compounds were detected as metabolites from dibenzofuran by the NarAaAb-expressing Rhodococcus strain, indicating that dibenzofuran was converted by lateral dioxygenase activity of NarA dioxygenase. Based on reverse transcriptase-polymerase chain reaction analysis, it was found that the narAaAb genes were cotranscribed and that their expression was induced in the presence of aromatic hydrocarbon compounds. It is likely that these genes are involved in the degradation pathways of a wide range of aromatic hydrocarbons by this strain. Strain SAO101 harbors three huge linear plasmids, pWK301 (1,100 kbp), pWK302 (1,000 kbp), and pWK303 (700 kbp), and the nar genes were found to be located on the pWK301 plasmid.

Drosophila melanogaster resistance against the parasitoid wasp Leptopilina boulardi is under the control of a single gene (Rlb), with two alleles, the resistant one being dominant. Using strains bearing deletions, we previously demonstrated that the 55E2-E6; 55F3 region on chromosome 2R is involved in the resistance phenomenon. In this paper, we first restricted the Rlb containing region by mapping at the molecular level the breakpoints of the Df(2R)Pc66, Df(2R)P34 and Df(2R)Pc4 deficiencies, using both chromosomal in situ hybridization and Southern analyses. The resistance gene was localized in a 100 kb fragment, predicted to contain about 10 different genes. Male recombination genetic experiments were then performed, leading to identification of two possible candidates for the Rlb gene. Potential involvement of one of this genes, edl/mae, is discussed.

A novel 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) gene of 1.35 kb was cloned from a cosmid library of Halomonas variabilis HTG7, inserted into vector pET-28a (+) and transformed in Escherichia coli BL21 (DE3). EPSPS was over-expressed in soluble form after induction with IPTG at 30 degrees C and it showed a single band in SDS-PAGE, which corresponds to a molecular weight of 51 kD. Deduced amino acid sequence analysis showed that there is little homology with the aroA genes which encode glyphosate-tolerant EPSPS in known sources, such as E. coli K12 and Agrobacterium sp. CP4. The over-expressed EPSPS was purified on nickel-nitrilotriacetic acid resin and detected by Western blotting analysis. Enzyme activity measurements demonstrated that there were 4.27 units/mg in cell extract, compared with 0.049 units/mg of the control. There is an 87-fold increase in specific activity for EPSPS.

The lipid A and core regions of the lipopolysaccharide in Rhizobium leguminosarum, a nitrogen-fixing plant endosymbiont, are strikingly different from those of Escherichia coli. In R. leguminosarum lipopolysaccharide, the inner core is modified with three galacturonic acid (GalA) moieties, two on the distal 3-deoxy-D-manno-octulosonic acid (Kdo) unit and one on the mannose residue. Here we describe the expression cloning of three novel GalA transferases from a 22-kb R. leguminosarum genomic DNA insert-containing cosmid (pSGAT). Two of these enzymes modify the substrate, Kdo2-[4'-(32)P]lipid IV(A) and its 1-dephosphorylated derivative on the distal Kdo residue, as indicated by mild acid hydrolysis. The third enzyme modifies the mannose unit of the substrate mannosyl-Kdo2-1-dephospho-[4'-(32)P]lipid IV(A). Sequencing of a 7-kb subclone derived from pSGAT revealed three putative membrane-bound glycosyltransferases, now designated RgtA, RgtB, and RgtC. Transfer by tri-parental mating of these genes into Sinorhizobium meliloti 1021, a strain that lacks these particular GalA residues, results in the heterologous expression of the GalA transferase activities seen in membranes of cells expressing pSGAT. Reconstitution experiments with the individual genes demonstrated that the activity of RgtA precedes and is necessary for the subsequent activity of RgtB, which is followed by the activity of RgtC. Electrospray ionization-tandem mass spectrometry and gas-liquid chromatography of the product generated in vitro by RgtA confirmed the presence of a GalA moiety. No in vitro activity was detected when RgtA was expressed in Escherichia coli unless Rhizobiaceae membranes were also included.

The acyl carrier proteins (ACPs) of fatty acid synthesis are functional only when modified by attachment of the prosthetic group, 4'-phosphopantetheine (4'-PP), which is transferred from CoA to the hydroxyl group of a specific serine residue. Almost 40 years ago Vagelos and Larrabee reported an enzyme from Escherichia coli that removed the prosthetic group. We report that this enzyme, called ACP hydrolyase or ACP phosphodiesterase, is encoded by a gene (yajB) of previously unknown function that we have renamed acpH. A mutant E. coli strain having a total deletion of the acpH gene has been constructed that grows normally, showing that phosphodiesterase activity is not essential for growth, although it is required for turnover of the ACP prosthetic group in vivo. ACP phosphodiesterase (AcpH) has been purified to homogeneity for the first time and is a soluble protein that very readily aggregates upon overexpression in vivo or concentration in vitro. The purified enzyme has been shown to cleave acyl-ACP species with acyl chains of 6-16 carbon atoms and is active on some, but not all, non-native ACP species tested. Possible physiological roles for AcpH are discussed.

Direct gene transfer into neurons has potential for both studying neuronal physiology and for developing gene therapy treatments for specific neurological conditions. Due to the heterogeneous cellular composition of the brain, cell-type-specific recombinant gene expression is required for many potential applications of neuronal gene transfer. The two prevalent approaches for achieving cell-type-specific expression are to use a cell-type-specific promoter to control recombinant gene expression or to modify a virus vector particle to target gene transfer to a specific cell type. Targeted gene transfer to multiple peripheral cell types has been described, but targeted gene transfer to a specific type of neuron in the brain has yet to be reported. Targeted gene transfer approaches with Herpes Simplex Virus (HSV-1) vectors have focused on modifying glycoprotein C (gC) to remove the heparin binding domain and add a binding activity for a specific protein on the cell surface. This study was designed to develop HSV-1 vectors that target gene transfer to cells that contain receptors for either glial-cell-line-derived neurotrophic factor (GDNF) or brain-derived neurotrophic factor (BDNF), such as nigrostriatal neurons. We isolated chimeric gC-GDNF or chimeric gC-BDNF constructs, and the resulting proteins were incorporated into HSV-1 virus particles. We performed helper virus-free HSV-1 vector packaging in the presence of each chimeric protein. The resulting vector stocks supported 2.2- to 5.0-fold targeted gene transfer to nigrostriatal neurons in the rat brain, compared to vector particles that contained wild-type (wt) gC. Gene transfer to nigrostriatal neurons by vector particles that contained chimeric gC-BDNF was reduced by preincubation with an anti-BDNF antibody. Targeted gene transfer to neurons that contain specific neurotrophic factor receptors may benefit specific physiological and gene therapy studies.

The T to C substitution at position -175 of the gamma-globin gene has been identified in some individuals with non-deletion hereditary persistence of fetal hemoglobin (HPFH). In this study, the HPFH phenotype was reestablished in transgenic mice carrying the mu'LCRAgamma(-175)psibetadeltabeta construct, which contained a 3.1-kb mu'LCR cassette linked to a 29-kb fragment from the Agamma-to beta-globin gene with the natural chromosome arrangement but with the -175 mutation, which provided evidence for this single mutation as the cause of this form of HPFH. The HPFH phenotype was also reproduced in transgenic mice carrying the mu'LCRAgamma(-173)psibetadeltabeta construct, in which the -175 T to C Agamma gene was substituted with the -173 T to C Agamma gene. In vitro experiments proved that the -175 mutation significantly reduced binding of Oct-1 but not GATA-1, whereas the -173 mutation dramatically decreased binding of GATA-1 but not Oct-1. These results suggest that abrogation of either GATA-1 or Oct-1 binding to this promoter region may result in the HPFH phenotype. An in vivo footprinting assay revealed that either the -175 mutation or the -173 mutation significantly decreased overall protein binding to this promoter region in adult erythrocytes of transgenic mice. We hypothesize that a multiprotein complex containing GATA-1, Oct-1, and other protein factors may contribute to the formation of a repressive chromatin structure that silences gamma-globin gene expression in normal adult erythrocytes. Both the -173 and -175 T to C substitutions may disrupt the complex assembly and result in the reactivation of the gamma-globin gene in adult erythrocytes.

Background: The pregnancy-specific glycoprotein (Psg) genes encode proteins of unknown function, and are members of the carcinoembryonic antigen (Cea) gene family, which is a member of the immunoglobulin gene (Ig) superfamily. In rodents and primates, but not in artiodactyls (even-toed ungulates / hoofed mammals), there have been independent expansions of the Psg gene family, with all members expressed exclusively in placental trophoblast cells. For the mouse Psg genes, we sought to determine the genomic organisation of the locus, the expression profiles of the various family members, and the evolution of exon structure, to attempt to reconstruct the evolutionary history of this locus, and to determine whether expansion of the gene family has been driven by selection for increased gene dosage, or diversification of function., Results: We collated the mouse Psg gene sequences currently in the public genome and expressed-sequence tag (EST) databases and used systematic BLAST searches to generate complete sequences for all known mouse Psg genes. We identified a novel family member, Psg31, which is similar to Psg30 but, uniquely amongst mouse Psg genes, has a duplicated N1 domain. We also identified a novel splice variant of Psg16 (bCEA). We show that Psg24 and Psg30 / Psg31 have independently undergone expansion of N-domain number. By mapping BAC, YAC and cosmid clones we described two clusters of Psg genes, which we linked and oriented using fluorescent in situ hybridisation (FISH). Comparison of our Psg locus map with the public mouse genome database indicates good agreement in overall structure and further elucidates gene order. Expression levels of Psg genes in placentas of different developmental stages revealed dramatic differences in the developmental expression profile of individual family members., Conclusion: We have combined existing information, and provide new information concerning the evolution of mouse Psg exon organization, the mouse Psg genomic locus structure, and the expression patterns of individual Psg genes. This information will facilitate functional studies of this complex gene family.

Different species of Leishmania are responsible for the diverse pathologies associated with leishmaniasis including Leishmania donovani which results in fatal visceral infection and Leishmania major which causes non-fatal cutaneous infection. In an attempt to identify genotypic differences between these related Old World Leishmania species which contribute to their distinct phenotypic characteristics, we have introduced a L. donovani cosmid library into L. major to select for L. donovani sequences which may increase L. major virulence in BALB/c mice. Through this approach, we have identified a region of the L. donovani genome which increased virulence in both visceral and cutaneous sites and was divergent from the corresponding region of the L. major genome. When these L. donovani sequences were reintroduced into L. major, they enhanced the overall virulence of L. major, increasing its ability to survive in both visceral and cutaneous sites. The region responsible for increased infection levels was determined to be the miniexon gene array derived from chromosome 36 of L. donovani. Pulse field electrophoresis revealed that L. donovani contained miniexon gene sequences in several chromosome locations as opposed to L. major which contains miniexon gene sequences only in chromosome 2. Because of the requirement for miniexon-derived transcripts in maturation of pre-mRNAs in trypanosomatids, this observation suggests that the increased expression of miniexon genes is associated with increased virulence. As the genome sequence for Leishmania becomes available, the in vivo selection procedure described within will be useful to identify additional species-specific sequences responsible for different pathogenic phenotypes associated with Leishmania infection.

We previously described a family of five RNA-binding proteins: CUG-binding protein, embryonic lethal abnormal vision-type RNA-binding protein 3, and the CUG-binding protein and embryonic lethal abnormal vision-type RNA-binding protein 3-like factors (CELFs) 3, 4, and 5. We demonstrated that all five of these proteins specifically activate exon inclusion of cardiac troponin T minigenes in vivo via muscle-specific splicing enhancer (MSE) sequences. We also predicted that a sixth family member, CELF6, was located on chromosome 15. Here, we describe the isolation and characterization of CELF6. Like the previously described CELF proteins, CELF6 shares a domain structure containing three RNA-binding domains and a divergent domain of unknown function. CELF6 is strongly expressed in kidney, brain, and testis and is expressed at very low levels in most other tissues. In the brain, expression is widespread and maintained from the fetus to the adult. CELF6 activates exon inclusion of a cardiac troponin T minigene in transient transfection assays in an MSE-dependent manner and can activate inclusion via multiple copies of a single element, MSE2. These results place CELF6 in a functional subfamily of CELF proteins that includes CELFs 3, 4, and 5. CELF6 also promotes skipping of exon 11 of insulin receptor, a known target of CELF activity that is expressed in kidney.

The MARK protein kinases were originally identified by their ability to phosphorylate a serine motif in the microtubule-binding domain of tau that is critical for microtubule binding. Here, we report the cloning and expression of a novel human paralog, MARK4, which shares 75% overall homology with MARK1-3 and is predominantly expressed in brain. Homology is most pronounced in the catalytic domain (90%), and MARK4 readily phosphorylates tau and the related microtubule-associated protein 2 (MAP2) and MAP4. In contrast to the three paralogs that all exhibit uniform cytoplasmic localization, MARK4 colocalizes with the centrosome and with microtubules in cultured cells. Overexpression of MARK4 causes thinning out of the microtubule network, concomitant with a reorganization of microtubules into bundles. In line with these findings, we show that a tandem affinity-purified MARK4 protein complex contains alpha-, beta-, and gamma-tubulin. In differentiated neuroblastoma cells, MARK4 is localized prominently at the tips of neurite-like processes. We suggest that although the four MARK/PAR-1 kinases might play multiple cellular roles in concert with different targets, MARK4 is likely to be directly involved in microtubule organization in neuronal cells and may contribute to the pathological phosphorylation of tau in Alzheimer's disease.

Fruiting body development in fungi is a complex cellular differentiation process that is controlled by more than 100 developmental genes. Mutants of the filamentous fungus Sordaria macrospora showing defects in fruiting body formation are pertinent sources for the identification of components of this multicellular differentiation process. Here we show that the sterile mutant pro11 carries a defect in the pro11 gene encoding a multimodular WD40 repeat protein. Complementation analysis indicates that the wild-type gene or C-terminally truncated versions of the wild-type protein are able to restore the fertile phenotype in mutant pro11. PRO11 shows significant homology to several vertebrate WD40 proteins, such as striatin and zinedin, which seem to be involved in Ca2+-dependent signaling in cells of the central nervous system and are supposed to function as scaffolding proteins linking signaling and eukaryotic endocytosis. Cloning of a mouse cDNA encoding striatin allowed functional substitution of the wild-type protein with restoration of fertility in mutant pro11. Our data strongly suggest that an evolutionarily conserved cellular process controlling eukaryotic cell differentiation may regulate fruiting body formation.

Type 2 diabetes mellitus (T2DM) is a group of multifactorial disorders due to either defective insulin secretion or action. Despite the fact that numerous genetic researches of T2DM have been pursued, the pathogenic mechanisms remain obscure. We encountered a T2DM family associated with a balanced reciprocal translocation, t(3;9)(p21.31;q33.1). To isolate a candidate gene susceptible to T2DM, we constructed physical maps covering both the 3p and 9q breakpoints of the translocation in the family. Consequently, the inositol hexaphosphate kinase 1 gene ( IHPK1) (OMIM *606991) was found to be disrupted at the 3p21.31 breakpoint. We then carried out sequence analysis for all coding regions of IHPK1 in 405 unrelated T2DM patients in order to validate whether aberrations of the gene are common in T2DM patients, but we failed to detect any pathogenic changes. The disruption of IHPK1 or another predisposing gene affected by position effect of the translocation may explain the T2DM phenotype at least in this family. Alternatively, the IHPK1 disruption in the family is a chance association.

We report the map-based cloning of the leaf rust resistance gene Lr21, previously mapped to a gene-rich region at the distal end of chromosome arm 1DS of bread wheat (Triticum aestivum L.). Molecular cloning of Lr21 was facilitated by diploid/polyploid shuttle mapping strategy. Cloning of Lr21 was confirmed by genetic transformation and by a stably inherited resistance phenotype in transgenic plants. Lr21 spans 4318 bp and encodes a 1080-amino-acid protein containing a conserved nucleotide-binding site (NBS) domain, 13 imperfect leucine-rich repeats (LRRs), and a unique 151-amino-acid sequence missing from known NBS-LRR proteins at the N terminus. Fine-structure genetic analysis at the Lr21 locus detected a noncrossover (recombination without exchange of flanking markers) within a 1415-bp region resulting from either a gene conversion tract of at least 191 bp or a double crossover. The successful map-based cloning approach as demonstrated here now opens the door for cloning of many crop-specific agronomic traits located in the gene-rich regions of bread wheat.

CDX1 is a homeobox protein that inhibits proliferation of intestinal epithelial cells and regulates intestine-specific genes involved in differentiation. CDX1 expression is developmentally and spatially regulated, and its expression is aberrantly down-regulated in colorectal cancers and colon cancer-derived cell lines. However, very little is known about the molecular mechanism underlying the regulation of CDX1 gene expression. In this study, we characterized the CDX1 gene structure and identified that its gene promoter contained a typical CpG island with a CpG observed/expected ratio of 0.80, suggesting that the CDX1 gene is a target of aberrant methylation. Alterations of DNA methylation in the CDX1 gene promoter were investigated in a series of colorectal cancer cell lines. Combined Bisulfite Restriction Analysis (COBRA) and bisulfite sequencing analysis revealed that the CDX1 promoter is methylated in CDX1 non-expressing colorectal cancer cell lines but not in human normal colon tissue and T84 cells, which express CDX1. Treatment with 5'-aza-2'-deoxycytidine (5-azaC), a DNA methyltransferase inhibitor, induced CDX1 expression in the colorectal cancer cell lines. Furthermore, de novo methylation was determined by establishing stably transfected clones of the CDX1 promoter in SW480 cells and demethylation by 5-azaC-activated reporter gene expression. These results indicate that aberrant methylation of the CpG island in the CDX1 promoter is one of the mechanisms that mediate CDX1 down-regulation in colorectal cancer cell lines.

Nick translation is used to label DNA and RNA to produce probes for in situ hybridization and Northern and Southern blotting. Fluorescence in situ hybridization (FISH) is a widely applied technique used to determine chromosomal and genetic anomalies in many biological samples. Initially the technique was applied to metaphase preparations, but the usefulness of detecting genetic anomalies in solid tumors in situ has resulted in the development of modified protocols. Formalin fixed paraffin processed tissue sections present novel challenges when applying FISH; the probes must be small (between 200 and 600 base pairs) and pretreatment is necessary before the probes can be applied to tissue sections, to promote probe access to target DNA. Here we report on a modification of a nick translation method to produce a probe that can reliably be used with FISH in paraffin processed tissue sections.

A hsp70-lacZ fusion gene introduced into Drosophila melanogaster at the euchromatic 31B region by Pelement transformation displayed a variegated expression with respect to the lacZ fusion protein in the salivary gland cells under heat-shock conditions. The variegation is also reflected by the chromosome puffing pattern. Subsequent transposition of the 31B P element to other euchromatic positions restored wild-type activity, that is, a nonvariegated phenotype. A lower developmental temperature reduced the amount of expression under heat-shock conditions, similar to genes undergoing position-effect variegation (PEV). However, other modifiers of PEV did not affect the expression pattern of the gene. These results show a novel euchromatic tissue-specific variegation that is not associated with classical heterochromatic PEV.

The anthracycline-like polyketide drug elloramycin is produced by Streptomyces olivaceus Tü2353. Elloramycin has antibacterial activity against Gram-positive bacteria and also exhibits antitumor activity. From a cosmid clone (cos16F4) containing part of the elloramycin biosynthesis gene cluster, three genes (elmMI, elmMII, and elmMIII) have been cloned. Sequence analysis and data base comparison showed that their deduced products resembled S-adenosylmethionine-dependent O-methyltransferases. The genes were individually expressed in Streptomyces albus and also coexpressed with genes involved in the biosynthesis of l-rhamnose, the 6-deoxysugar attached to the elloramycin aglycon. The resulting recombinant strains were used to biotransform three different elloramycin-type compounds: l-rhamnosyl-tetracenomycin C, l-olivosyl-tetracenomycin C, and l-oleandrosyl-tetracenomycin, which differ in their 2'-, 3'-, and 4'-substituents of the sugar moieties. When only the three methyltransferase-encoding genes elmMI, elmMII, and elmMIII were individually expressed in S. albus, the methylating activity of the three methyltransferases was also assayed in vitro using various externally added glycosylated substrates. From the combined results of all of these experiments, it is proposed that methyltransferases ElmMI, ElmMII, and ElmMIII are involved in the biosynthesis of the permethylated l-rhamnose moiety of elloramycin. ElmMI, ElmMII, and ElmMIII are responsible for the consecutive methylation of the hydroxy groups at the 2'-, 3'-, and 4'-position, respectively, after the sugar moiety has been attached to the aglycon.

The protective barrier provided by stratified squamous epithelia relies on the cornified cell envelope (CE), a structure synthesized at late stages of keratinocyte differentiation. It is composed of structural proteins, including involucrin, loricrin, and the small proline-rich (SPRR) proteins, all encoded by genes localized at human chromosome 1q21. The genetic characterization of the SPRR locus reveals that the various members of this multigene family can be classified into two distinct groups with separate evolutionary histories. Whereas group 1 genes have diverged in protein structure and are composed of three different classes (SPRR1 (2x), SPRR3, and SPRR4), an active process of gene conversion has counteracted diversification of the protein sequences of group 2 genes (SPRR2 class, seven genes). Contrasting with this homogenization process, all individual members of the SPRR gene family show specific in vivo and in vitro expression patterns and react selectively to UV irradiation. Apparently, creation of regulatory rather than structural diversity has been the driving force behind the evolution of the SPRR gene family. Differential regulation of highly homologous genes underlines the importance of SPRR protein dosage in providing optimal barrier function to different epithelia, while allowing adaptation to diverse external insults.

Herpes simplex virus type 1 (HSV-1) amplicons are prokaryotic plasmids containing one or more transcriptional units and two cis-acting HSV-1 sequences: a viral origin of DNA replication and a viral DNA cleavage/packaging signal. In the presence of HSV-1 "helper" functions, amplicons are replicated and packaged into HSV-1 virions. Despite recent improvements in packaging methods, stocks of amplicon vectors are still contaminated with replication-competent helper virus at a frequency of 10(-4)-10(-6). To overcome this problem, we report that: (i) genetic modifications of HSV-1 genomes can be routinely achieved in Escherichia coli, either by homologous or site-specific recombination, (ii) a novel HSV-1 bacterial artificial chromosome (fHSVDeltapacDelta27 0+), which has a deletion in the essential gene encoding ICP27 and an addition of ICP0 "stuffer" sequences to increase its size to 178 kb, supports the replication and packaging of cotransfected amplicon DNA without generating replication-competent helper virus (<1 helper virus per 10(8) TU amplicon vectors), and (iii) the resulting amplicon stocks have titers of up to 3-10 x 10(8) TU/ml after concentration. Elimination of replication-competent helper virus from HSV-1 amplicon vector stocks further improves safety in gene transfer applications.

vvd, a gene regulating light responses in Neurospora, encodes a novel member of the PAS/LOV protein superfamily. VVD defines a circadian clock-associated autoregulatory feedback loop that influences light resetting, modulates circadian gating of input by connecting output and input, and regulates light adaptation. Rapidly light induced, vvd is an early repressor of light-regulated processes. Further, vvd is clock controlled; the clock gates light induction of vvd and the clock gene frq so identical signals yield greater induction in the morning. Mutation of vvd severely dampens gating, especially of frq, consistent with VVD modulating gating and phasing light-resetting responses. vvd null strains display distinct alterations in the phase-response curve to light. Thus VVD, although not part of the clock, contributes significantly to regulation within the Neurospora circadian system.

The linear plasmid SCP1 is integrated into the central region of the chromosome of Streptomyces coelicolor A3(2). The integrated structures of SCP1 in two bidirectional donor strains, 2612 and A634, were analyzed by cloning and sequencing of the junctions between the SCP1 DNA and the chromosomal DNA. In the NF (normal fertility) strain 2612, SCP1 is integrated in a right-handed direction into ORF-X at the left end of the IS cluster in AseI fragment E. An almost intact left end of SCP1 is retained, while the right terminal inverted repeat (TIR-R) of SCP1 and a 33-kb chromosomal DNA segment including the IS cluster are deleted. In the NF-like strain A634, SCPI is also integrated into AseI fragment E in a left-handed direction. The left junction is composed of IS466 with complete deletion of TIR-R of SCP1, and the right junction is located at the left end of IS468A* with half of TIR-L being deleted. During the integration event, a 5.4-kb chromosomal DNA segment including IS468A, IS468B, IS469 and IS466A was duplicated so that this sequence is now present on both sides of SCP1. Since 2612 and A634 exhibit a similar bidirectional gradient of gene transfer, it is surprising that their chromosomal structures are so different.

We describe a procedure for recycling nylon hybridization membranes, enabling their repeated use for radioactive Southern hybridization analysis of different DNA samples. Following hybridization and probe removal, nylon membranes containing covalently linked DNAs were treated with 0.55% sodium hypochlorite. This destroyed the DNA, thereby preventing it from participating in further hybridization and enabling the membranes to be used subsequently for binding new DNA samples. With this procedure, we were able to reuse a single membrane as many as 13 times, with no detectable loss in signal. This method was shown to be effective for membranes supplied by three different manufacturers.

Previous studies of cloned ribosomal DNA (rDNA) variants isolated from the cosmid library of human chromosome 13 have revealed some disproportion in representativity of different rDNA regions (N. S. Kupriyanova, K. K. Netchvolodov, P. M. Kirilenko, B. I. Kapanadze, N. K. Yankovsky, and A. P. Ryskov, Mol. Biol. 30, 51-60, 1996). Here we show nonrandom cleavage of human rDNA with Sau3A or its isoshizomer MboI under mild hydrolysis conditions. The hypersensitive cleavage sites were found to be located in the ribosomal intergenic spacer (rIGS), especially in the regions of about 5-5.5 and 11 kb upstream of the rRNA transcription start point. This finding is based on sequencing mapping of the rDNA insert ends in randomly selected cosmid clones of human chromosome 13 and on the data of digestion kinetics of cloned and noncloned human genomic rDNA with Sau3A and MboI. The results show that a methylation status and superhelicity state of the rIGS have no effect on cleavage site sensitivity. It is interesting that all primary cleavage sites are adjacent to or entering into Alu or Psi cdc 27 retroposons of the rIGS suggesting a possible role of neighboring sequences in nuclease accessibility. The results explain nonequal representation of rDNA sequences in the human genomic DNA library used for this study., (Copyright 2000 Academic Press.)

A helper virus-free herpes simplex virus type-1 (HSV-1) plasmid vector system developed recently may have applications in gene therapy and basic physiological studies. This system might be improved by mutating specific HSV-1 genes in the packaging system and by creating large vectors. An in vivo recombination cloning procedure is reported that supports the routine manipulation of relatively large DNAs such as the five cosmids that comprise this helper virus-free HSV-1 packaging system. In vivo recombination cloning is carried out by transforming overlapping DNA fragments into a specific RecA+ Escherichia coli, BJ5183. The cloning efficiency was improved by using a modified version of the Hanahan transformation procedure, and the background was lowered by either using vectors with different combinations of ends (5' overhangs, 3' overhangs, blunt ends) or by treating the vector with calf intestinal phosphatase. The range of usable overlap sizes is from 251 bp to 18 kb with 500 bp to 5 kb preferred. This procedure supports the routine construction and mutation of HSV-1 cosmids, by use of up to six different DNA fragments, and the construction of plasmids up to 65 kb in size. This procedure may also have applications to other vector systems and to studies on large viruses.

To perform the complex manipulation of the adenoviral genome for the construction of recombinant adenovirus vectors, we developed a cosmid vector (pacad1A) from which an infectious E1 and E3-deleted adenoviral genome can be released with PacI digestion. The cosmid vector, pacad1A, has unique restriction enzyme sites that are created for the insertion of foreign genes into the deleted E1 or E3 region of the adenoviral genome. To demonstrate the feasibility of the construction of adenovirus vectors with our developed vector, we showed that a recombinant adenovirus bearing a self-contained tetracycline-regulated expression system could be generated by transfection of cells with an infectious adenoviral genome that was released from pacad1A-derived plasmid DNA. The recombinant adenovirus vector was obtained easily by this method, and the expression of a transgene was proved to be regulated with tetracycline in CHO-K1 cells.

Surface-exposed unusual lipids containing phthiocerol and phenolphthiocerol are found only in the cell wall of slow-growing pathogenic mycobacteria and are thought to play important roles in host-pathogen interaction. The enzymology and molecular genetics of biosynthesis of phthiocerol and phenolphthiocerol are unknown. We postulate the domain organization of a set of multifunctional enzymes and a cluster of genes (pps) that would encode these enzymes for the biosynthesis of phthiocerol and phenolphthiocerol. A cosmid containing the postulated pps gene cluster was identified by screening a genomic library of Mycobacterium bovis BCG with the postulated homologous domains from mycocerosic acid synthase and fatty acid synthase genes as probes. Homologous cosmids were also identified in the genomic libraries of Mycobacterium tuberculosis and Mycobacterium leprae. M. bovis BCG was transformed with a pps disruption construct, made from the BCG cosmid by introducing the hygromycin resistance gene as the positive-selectable marker and the sacB gene as the counter-selectable marker. Gene disruption by homologous recombination with double crossover was confirmed by polymerase chain reaction and Southern hybridization. Chromatographic analysis showed that the phenolphthiocerol derivative, mycoside B, and phthiocerol dimycocerosates were not produced by the gene knockout mutants. This result confirms the identity of the pps genes. With the identification of the pps gene clusters in both M. tuberculosis and M. leprae, it should be possible to test the postulated roles of these unique lipids in tuberculosis and leprosy.

In this paper, we describe our algorithmic approach to constructing ordered restriction maps based on the data created from the images of population of individual DNA molecules (clones) digested by restriction enzymes. The goal is to devise map-making algorithms capable of producing high-resolution, high-accuracy maps rapidly and in a scalable manner. The resulting software is a key component of our optical mapping automation tools and has been used routinely to map cosmid, lambda and BAC clones. The experimental results appear highly promising.

To study the fate of linear DNA in Escherichia coli cells, we linearized plasmid DNA at a specific site in vivo and monitored its behavior in recA mutant cells deficient in recombinational repair. Earlier, we had found that in wild-type (WT) cells linearized DNA is degraded to completion by RecBCD nuclease. We had also found that in WT cells chi sites on linear DNA inhibit RecBCD degradation by turning off its nucleolytic activities. Now we report that chi sites do not work in the absence of the RecA protein, suggesting that RecA is required in vivo to turn off the degradative activities of the RecBCD enzyme. We also report that the degradation of linearized plasmid DNA, even devoid of chi sites, is never complete in recA cells. Investigation of this linear DNA stability indicates that a fraction of recA cells are recBC phenocopies due to ongoing chromosomal DNA degradation, which titrates RecBCD nuclease. A possible role for RecBCD-promoted DNA degradation in controlling chromosomal DNA replication in E. coli is discussed.

We have constructed a detailed map of the genomic region containing the ETS-variant gene 6 (ETV6), involved in translocations and deletions associated with hematologic malignancies. Thirty-eight cosmids were characterized belonging to two contigs spanning 340 kb, and an EcoRl restriction map was developed. The gap between the two contigs, 2 kb in size, was closed by PCR. The contigs contain the complete coding sequence and the 5' and 3' UTRs of ETV6. Eight exons accounting for the ETV6 cDNA sequence were identified. The helix-loop-helix (HLH) motif is coded by exons 3 and 4, whereas exons 6-8 code for the ETS DNA-binding domain. All introns show consensus 5' donor and 3' acceptor splice sites. Introns 1 and 2 span 100 and 82 kb, respectively, and introns 3-7 range from 15 to 1.3 kb. An alternative exon 1 (exon 1B) is localized in intron 2. The 5' end of the ETV6 gene is associated with a CpG island characterized by the presence of four Notl, four Sacll, and three BssHll recognition sites and several SP1- and AP2-binding motifs. Alternative polyadenylation at the 3' end of the ETV6 gene generates the three transcripts of 6200, 4300, and 2400 nucleotides, respectively. The ETV6 gene spans 240 kb and is flanked at its 5' and 3' end by D12S1697 and D12S98, respectively. The markers D12S1095 and D12S89 are located in the first intron. Two new DNA polymorphisms were identified in the ETV6 gene, which will be useful for the analysis of loss of heterozygosity reported for the ETV6 gene in leukemia.

In Escherichia coli, unprotected linear DNA is degraded by exoV activity of the RecBCD nuclease, a protein that plays a central role in the repair of double-strand breaks. Specific short asymmetric sequences, called chi sites, are hotspots for RecBCD-promoted recombination and are shown in vitro to attenuate exoV activity. To study RecBCD-chi site interactions in vivo we used phage lambda's terminase to introduce a site-specific double-strand break at lambda's cos site inserted into a plasmid. We show that after terminase has cut cos in vivo, nucleases degrade linearized DNA only from the end that does not have a strong terminase binding site. Linearized cosmid DNA containing chi sites in the proper orientation to the unprotected end is degraded more slowly in rec+ E. coli than is chi-less DNA. Increased survival of chi-containing DNA is a result of partial inactivation of exoV activity and is dependent on RecA and SSB proteins. The linearization of chi-containing DNA molecules leads to RecA-dependent formation of branched structures which have been proposed as intermediates in the RecBCD pathway of double-strand break repair.