Expression of bacterial cysteine biosynthesis genes in transgenic mice and sheep: toward a newin vivo amino acid biosynthesis pathway and improved wool growth

It is possible to improve wool growth through increasing the supply of cysteine available for protein synthesis and cell division in the wool follicle. As mammals can only synthesis cysteine indirectly from methionine via trans-sulphuration, expression of transgenes encoding microbial cysteine biosynthesis enzymes could provide a more efficient pathway to cysteine synthesis in the sheep. If expressed in the rumen epithelium, the abundant sulphide, produced by ruminal microorganisms and normally excreted, could be captured for conversion to cysteine. This paper describes the characterisation of expression of the cysteine biosynthesis genes ofSalmonella typhimurium, cysE,cysM andcysK, and linkedcysEM,cysME andcysKE genes as transgenes in mice and sheep. The linked transgenes were constructed with each gene driven by a separate promoter, either with the Rous sarcoma virus long terminal repeat (RSVLTR) promoter or the mouse phosphoglycerate kinase-1 (mPgk-1) promoter, and with human growth hormone (hGH) polyadenylation sequences. Transgenesis of mice with the RSVLTR-cysE gene afforded tissue-specific, heritable expression of the gene. Despite high levels of expression in a number of tissues, extremely low levels of expression occurred in the stomach and small intestine. Results of a concurrent sheep transgenesis experiment using the RSVLTR-cysEM and-cysME linked transgenes revealed that the RSVLTR promoter was inadequate for expression in the rumen. Moreover, instability of transgenes containing the RSVLTR sequence was observed. Expression of mPgk-cysME and-cysKE linked transgenes in most tissues of the mice examined, including the stomach and small intestine, suggested this promoter to be a better candidate for expression of these transgenes in the analogous tissues of sheep. However, a subsequent sheep transgenesis experiment indicated that use of the mPgk-1 promoter, active ubiquitously and early in development, may be inappropriate for expression of the cysteine biosynthesis transgenes. In summary, these results indicate that enzymically active bacterial cysteine biosynthesis gene products can be coexpressed in mammalian cellsin vivo but that expression of the genes should be spatio-temporally restricted to the adult sheep rumen epithelium.     

Molecular marker analyses of powdery mildew resistance in barley

Powdery mildew, caused byEryisphe graminis f. sp.hordei, is one of the most important diseases of barley (Hordeum vulgare). A number of loci conditioning resistance to this disease have been reported previously. The objective of this study was to use molecular markers to identify chromosomal regions containing genes for powdery mildew resistance and to estimate the resistance effect of each locus. A set of 28 F₁ hybrids and eight parental lines from a barley diallel study was inoculated with each of five isolates ofE. graminis. The parents were surveyed for restriction fragment length polymorphisms (RFLPs) at 84 marker loci that cover about 1100 cM of the barley genome. The RFLP genotypes of the F₁s were deduced from those of the parents. A total of 27 loci, distributed on six of the seven barley chromosomes, detected significant resistance effects to at least one of the five isolates. Almost all the chromosomal regions previously reported to carry genes for powdery mildew resistance were detected, plus the possible existence of 1 additional locus on chromosome 7. The analysis indicated that additive genetic effects are the most important component in conditioning powdery mildew resistance. However, there is also a considerable amount of dominance effects at most loci, and even overdominance is likely to be present at a number of loci. These results suggest that quantitative differences are likely to exist among alleles even at loci which are considered to carry major genes for resistance, and minor effects may be prevalent in cultivars that are not known to carry major genes for resistance.     

Mutation and cloning of clustered Streptomyces genes essential for sulphate metabolism

Summary A range of mutants auxotrophic for cysteine (cys) and resistant to selenate (sel) were isolated from many Streptomyces strains but chiefly from S. coelicolor A3(2) and S. lividans 66. Two of the classes of sel/cys mutants probably contained simple biochemical lesions of sulphate permease (selC) and ATP sulphurylase (selA) activities, while a further two classes (selD and selE) were pleiotropic and possibly regulatory. Most classes of sel mutations were clustered around the cysD locus of S. coelicolor. Segments of chromosomal DNA cloned from S. coelicolor, S. cattleya and S. clavuligerus and able to complement various sel/cys mutations allowed the relative positions of these mutations and the cysC and cysD mutations of S. coelicolor to be determined. The sel/cys DNA can be used for two-way selection: Cys⁺Sel^sCys^-Sel^r.     

The genetics of some polymorphic Esterases in Drosophila simulans

Analysis of strains of Drosophila simulans derived from a natural population in Turkey has shown that three alleles at the Esterase-6 locus are present in some strains. These alleles produce allozymes with mobilities equivalent to other esterases coded for by three other loci, one of which () is tightly linked to Esterase-6. Active alleles at all these loci are present at low frequencies. The presence of the active allele at the locus in laboratory strains in absolutely correlated with the presence of the Esterase 6 ^-3 allele and strong linkage disequilibrium between these two loci exists.It appears possible that the origin of one or more of these hitherto unremarked loci, whose active alleles are expressed only in females, lies in gene duplication, coupled with the action of selection in an environment thought to be heavily     

Genes affecting the major outer membrane proteins of Escherichia coli K-12: Mutations at nmpA and nmpB

Summary Strains of Escherichia coli K-12 carrying mutations at either of two distinct loci (nmpA and nmpB) produce a new outer membrane pore protein which is not present in wild type cells. Mutations at either of these loci result in sensitivity to phage TC45, which can use this new protein as its receptor, and the new protein (the NmpAB protein) appears to be identical in both NmpA and NmpB mutants. In order to determine whether both of these loci contain structural genes for the NmpAB protein, strains carrying secondary mutations at either of these loci which produced altered proteins were sought by selecting for resistance to phage TC45. Mutants which produced proteins with altered electrophoretic mobility and altered peptide maps were isolated from strains carrying both nmpA and nmpB mutations, and these secondary mutations mapped at the same sites as the original mutations leading to production of the NmpAB protein. This suggests that both loci contain structural genes. Strains mutant at nmpB which can no longer produce the protein can mutate at the nmpA locus to produce the new protein, indicating that both genes can exist in the same cell. Since the altered proteins of mutant strains could be distinguished from one another, we attempted to construct strains in which both nmpA and nmpB were expressed. In all cases only the protein produced by the nmpB mutation was produced, indicating some form of cooperative regulation of the two genes.     

Identification and physical characterization of yeast maltase structural genes

Summary Each of at least five unlinked MAL loci (MAL1 through MAL4 and MAL6) on the yeast genome controls the ability to synthesize an inducible -D-glucosidase (maltase). A subcloned fragment of the coding sequence of the MAL6 maltase structural gene was used as a hybridization probe to investigate the physical structure of the family of MAL structural genes in the genomes of different Saccharomyces strains. Mal⁺ strains, each carrying a genetically defined MAL locus, were crossed with a Mal^- strain and the segregation behavior of the functional locus and of sequences complementary to the maltase structural gene at that locus analyzed. The maltase structural gene sequences of each MAL locus were detected by Southern blot hybridization using BamH1 digests of genomic DNA of the meiotic products. This restriction enzyme was previously shown to cleave outside the confines of the MAL6 locus.The results of such experiments indicate that each MAL locus encompasses at least one maltase structural gene sequence homologous to that of MAL6, that yeast strains that lack functional MAL loci may or may not contain the corresponding maltase structural gene sequence, that the MAL1 maltase structural gene sequence or one of its alleles can be detected in all laboratory yeast strains examined and that each MAL locus can be identified as a characteristic BamH1 fragment of genomic DNA which includes a maltase structural gene.Yeast strains vary in the number of maltase structural gene sequences that they carry. By using the approach described in this report, the ones corresponding to the different functional MAL loci and residing within a BamH1 generated restriction fragment can be identified.     

Identification of genetic determinants of IGF‐1 levels and longevity among mouse inbred strains

The IGF‐1 signaling pathway plays an important role in regulating longevity. To identify the genetic loci and genes that regulate plasma IGF‐1 levels, we intercrossed MRL/MpJ and SM/J, inbred mouse strains that differ in IGF‐1 levels. Quantitative trait loci (QTL) analysis of IGF‐1 levels of these F2 mice detected four QTL on chromosomes (Chrs) 9 (48 Mb), 10 (86 Mb), 15 (18 Mb), and 17 (85 Mb). Haplotype association mapping of IGF‐1 levels in 28 domesticated inbred strains identified three suggestive loci in females on Chrs 2 (13 Mb), 10 (88 Mb), and 17 (28 Mb) and in four males on Chrs 1 (159 Mb), 3 (52 and 58 Mb), and 16 (74 Mb). Except for the QTL on Chr 9 and 16, all loci co‐localized with IGF‐1 QTL previously identified in other mouse crosses. The most significant locus was the QTL on Chr 10, which contains the Igf1 gene and which had a LOD score of 31.8. Haplotype analysis among 28 domesticated inbred strains revealed a major QTL on Chr 10 overlapping with the QTL identified in the F2 mice. This locus showed three major haplotypes; strains with haplotype 1 had significantly lower plasma IGF‐1 and extended longevity (P < 0.05) than strains with haplotype 2 or 3. Bioinformatic analysis, combined with sequencing and expression studies, showed that Igf1 is the most likely QTL gene, but that other genes may also play a role in this strong QTL.     

Genetics of dietary obesity in AKR/JxSWR/J mice: segregation of the trait and identification of a linked locus on Chromosome 4

We describe a new multiple gene mouse model of differential sensitivity to dietary obesity that provides a tool for dissecting the genetic basis for body composition and obesity. AKR/J and SWR/J male mice, as well as male progeny of intercrosses between these strains, were fed a high-fat diet for 12 weeks beginning at 5 weeks of age. Body weight and energy intake were assessed weekly. At the conclusion of the dietary manipulation, an adiposity index was calculated by dividing the weight of seven dissected adipose depots by the carcass weight. AKR/J mice had approximately sixfold greater adiposity than SWR/J mice. Examination of the segregation of the adiposity trait in the progeny of crosses between these strains indicates that the trait is determined by a minimum of one to four genetic loci and that there is significant dominance of the AKR/J genotype. A preliminary analysis with markers linked to the known mouse obesity genes ob, db, tub, and fat showed no linkage with these loci. However, a quantitative trait locus was found that maps distal to the db gene on Chromosome (Chr) 4. This locus has been designated dietary obese 1 or Do1.     

Analysis of N-acyl homoserine-lactone quorum-sensing molecules made by different strains and biovars of Rhizobium leguminosarum containing different symbiotic plasmids

Strains of Rhizobium leguminosarum use a cell density-dependent gene regulatory system to assess their population density. This is achieved by the accumulation of N-acyl-homoserine lactones (AHLs) in the environment during growth of the bacteria and these AHLs stimulate the induction of various bacterial genes that are up-regulated in the late-exponential and stationary phases of growth. A genetically well-characterised strain of R. leguminosarum biovar viciae was found to have four genes, whose products synthesise different AHLs. We have analysed AHL production by four genetically distinct isolates of R. leguminosarum, three of bv. viciae and one of bv. phaseoli. Distinct differences were seen in the pattern of AHLs produced by the bv. viciae strains compared with bv. phaseoli and the increased levels and diversity of AHLs found in bv. viciae strains can be attributed to the rhiI gene, which is located on the symbiotic (Sym) plasmid and is up-regulated when the bacteria are grown in the rhizosphere. Additional complexity to the profile of AHLs is found to be associated with highly transmissible plasmid pRL1JI of R. leguminosarum bv. viciae, but this is not observed with some other strains, including those carrying different transmissible plasmids. In addition to AHLs produced by the products of genes on the symbiotic plasmid, there is clear evidence for the presence of other AHL production loci. Expression levels and patterns of AHLs can change markedly in different growth media. These results indicate that there is a network of quorum-sensing loci in different strains of R. leguminosarum and these loci may play a role in adapting to rhizosphere growth and plasmid transfer.     

Biochemical analysis of mutants defective in nitrate assimilation in Neurospora crassa: evidence for autogenous control by nitrate reductase

Summary A biochemical analysis of mutants altered for nitrate assimilation in Neurospora crassa is described. Mutant alleles at each of the nine nit (nitrate-nonutilizing) loci were assayed for nitrate reductase activity, for three partial activities of nitrate reductase, and for nitrite reductase activity. In each case, the enzyme deficiency was consistent with data obtained from growth tests and complementation tests in previous studies. The mutant strains at these nit loci were also examined for altered regulation of enzyme synthesis. Such exeriments revealed that mutations which affect the structural integrity of the native nitrate reductase molecule can result in constitutive synthesis of this enzyme protein and of nitrite reductase. These results provide very strong evidence that, as in Aspergillus nidulans, nitrate reductase autogenously regulates the pathway of nitrate assimilation. However, only mutants at the nit-2 locus affect the regulation of this pathway by nitrogen metabolite repression.     

Molecular population genetic analysis of the enn subdivision of group A streptococcal emm-like genes: horizontal gene transfer and restricted variation among enn genes

The group A streptococcal emm-like genes, which encode the cell-surface M and M-like proteins, are divided into distinct mrp, emm and enn subdivisions and are clustered together in a region of the chromosome called the vir regulon. In order to understand the mechanisms involved in the evolution of emm-like genes, a 180bp fragment of the 5 variable region of the enn gene was characterized in 31 strains for which emm sequences and multilocus enzyme electrophoretic profiles have been previously determined. The results demonstrate that nucleotide polymorphisms at the enn locus are generated predominantly by point mutations and short deletions or insertions, and that variation among enn and emm genes has arisen by similar mechanisms. However, diversity at the enn locus is restricted in comparison to the emm locus. Moreover, there is strong evidence for intragenic recombination at the enn locus and the pattern of distribution of emm and enn alleles among strains suggests that these genes may be independently acquired by horizontal transfer and recombination from distinct donor strains, thereby generating a mosaic structure for the vir regulon. The results add to a growing body of evidence that horizontal gene transfer has played a major role in the evolution of Streptococcus pyogenes vir regulons.     

Identification and physical characterization of yeast glucoamylase structural genes

Summary Each one of at least three unlinked STA loci (STA1, STA2 and STA3), in the genome of Saccharomyces diastaticus controls starch hydrolysis by coding for an extracellular glucoamylase. Cloned STA2 sequences were used as hybridization probes to investigate the physical structure of the family of STA genes in the genomes of different Saccharomyces strains. Sta⁺ strains, each carrying a single genetically defined STA locus, were crossed with a Sta^– strain and the segregation behavior of the functional locus (i.e. Sta⁺) and sequences homologous to a cloned STA2 glucoamylase structural gene at that locus were analyzed. The results indicate that in all strains examined there is a multiplicity of sequences that are homologous to STA2 DNA but that only the functional STA loci contain extensive 5 and 3 homology to each other and can be identified as residing on unique fragments of DNA; that all laboratory yeast strains examined contain extensive regions of the glucoamylase gene sequences at or closely linked to the STA1 chromosomal position; that the STA1 locus contains two distinct glucoamylase gene sequences that are closely linked to each other; and that all laboratory strains examined also contain another ubiquitous sequence that is not allelic to STA1 and is nonfunctional (Sta^–), but has retained extensive sequence homology to the 5 end of the cloned STA2 gene. It was also determined that the DEX genes (which control dextrin hydrolysis in S. diastaticus), MAL5 (a gene once thought to control maltose metabolism in yeast) and the STA genes are allelic to each other in the following manner: STA1 and DEX2, STA1 and MAL5, and STA2 and DEX1 and STA3 and DEX3.     

Identification of Escherichia coli genes whose expression increases as a function of external pH

Summary Using transposon TnphoA and a plate screening method, we have isolated a set of Escherichia coli strains carrying phoA fusions with genes whose expression is modulated as a function of external pH. Besides fusions with the ompF gene and the malB locus, thirteen independent fusions were analysed whose expression is maximal during growth at pHs ranging from 7.0 to 8.5 and minimal during growth at pH 5.0. Six different genetic loci, called phmA, phmB, phmC, phmD, phmE and phmF (for pH modulated) were characterized and localized on the E. coli chromosome at approx. 12, 18, 41, 45, 75 and 84 min, respectively. Expression of phmA: :phoA fusions is also influenced when internal pH or environmental conditions such as osmolarity or anaerobiosis are modified. EnvZ protein is not involved in the regulation of phm : :phoA fusions.     

Structure of the multigene family of MAL loci in Saccharomyces

Summary Multigene families are a ubiquitous feature of eukaryotes; however, their presence in Saccharomyces is more limited. The MAL multigene family is comprised of five unlined loci, MAL1, MAL2, MAL3, MAL4 and MAL6, any one of which is sufficient for yeast to metabolize maltose. A cloned MAL6 locus was used as a probe to facilitate the cloning of the other four functional loci as well as two partially active alleles of MAL1. Each locus could be characterized as a cluster of three genes, MALR (regulatory), MALT (maltose transport or permease) and MALS (structural or maltase), encoded by a total of about 7 kb of DNA; however, homologous sequences at each locus extend beyond the coding regions. Our results indicate that there is extensive homology among the MAL loci, especially within their maltase genes. The greatest sequence diversity occurs in their regulatory gene regions. Southern cross analyses of the cloned MAL loci indicate a single duplication of the MAL6R-homologous sequences upstream of the MAL6R gene as well as an extensive duplication of more than 10 kb at the MAL3 locus. The large repeat at the MAL3 locus results in the presence of four copies of MAL3R-homologous sequences and two copies of MAL3T-homologous sequences at that locus. Two naturally occurring inactive alleles of MAL1 show a deletion or divergence of their MALR sequences. The significance of these repeats in the evolution of the MAL multigene family is discussed.     

Evidence against mutational "hot-spots" at aggregation loci in Dictyostelium discoideum

Summary Complementation tests were performed on 10 strains of Dictyostelium discoideum which carry developmental mutations representing aggregation loci identified previously in two independent studies. When the 5 aggregation-deficient strains representing loci CG1-5 were fused with the 5 strains carrying mutations at loci agoA-E, all 25 crosses produced aggregation-competent diploids. Complicating factors, such as negative gene interactions and possible interallelic complementation are discussed. The results of this experiment suggest that the 10 aggregation loci identified in the two studies are different and that aggregation loci in D. discoideum are probably not associated with significant mutational hot-spots.     

Mapping and characterization of the DQ subregion of the ovine MHC

A map of the ovine MHC class II DQ subregion has been constructed from overlapping cosmid clones. This region consists of two loci linked on a linear tract of 130 kb DNA. Each locus consists of a DQA and a DQB gene in a tail-to-tail orientation. The genes in each locus are transcribed but only those designated DQ1 express class II molecules at the surface of mouse L cells following DNA-mediated gene transfection. The DQA1 and DQB1 genes are separated by 11kb while the DQA2 and B2 genes are 25 kb apart. The loci are separated by 22 kb.     

A genetic assay for transversion mutations in bacteriophage T4

Summary Seventy-two mutants deficient in formate-nitrate reductase activity were selected in Escherichia coli strain PK 27, by two different procedures. Forty-five strains were selected on the basis of chlorate resistance and 27 strains were selected by their inability to reduce nitrate with formate as an electron donor. Genetic analysis of these strains showed that the two techniques yield distinctly different distributions of mutants among the various controlling genetic loci. Chlorate resistance appears to select for severe alterations in the nitrate reductase system; 98% of these mutants fell into the pleiotropic chl A, B, D and E classes and are deficient in all the activities of the formate-hydrogenlyase pathway as well as formate-nitrate reductase pathway. In contrast, 48% of the mutants selected for their inability to reduce nitrate with formate as the electron donor were of the chl C class and two new classes were identified among mutants selected by this procedure. Chl F mutants are linked to tryptophan and the chl C locus. Chl G mutants map at zero minutes on the E. coli genetic map.     

Hydrogen sulfide formation as well as ethanol production in different media by cysND- and/or cysIJ-inactivated mutant strains of Zymomonas mobilis ZM4

Many bacteria reduce inorganic sulfate to sulfide to satisfy their need for sulfur, one of the most important elements for biological life. But little is known about the metabolic pathways involving hydrogen sulfide (H₂S) in mesophilic bacteria. By genomic sequence analysis, a complete set of genes for the assimilatory sulfate reduction pathway has been identified in the ethanologen Zymomonas mobilis. In this study, the first ATP sulfurylase- and final sulfite reductase-encoding genes cysND and cysIJ, respectively, in the putative pathway from sulfate to sulfite in Z. mobilis ZM4 was singly or doubly inactivated by homologous recombination and a site-specific FLP-FRT recombination. The resultant mutants, ?cysND, ?cysIJ and ?cysND-cat?cysIJ, were unable to produce detectable H₂S in glucose or sucrose-containing rich medium and sweet sorghum juice, in which the wild-type ZM4 produced detectable H₂S. While adding sulfite (SO₃ ^2?) into media impaired the growth of the mutants and ZM4 to varying degrees, the sulfite restored the H₂S formation in the ?cysND in the above media, but not in the ?cysIJ and ?cysND-cat?cysIJ mutants. Although it seemed that the inactivation of cysND and cysIJ did not exert a significant negative effect on the cell growth at least in glucose or sucrose medium, the ethanol production of all mutants was inferior to that of ZM4 in sucrose medium and sweet sorghum juice. In addition, adding l-cysteine to glucose-containing rich media restored H₂S formation of all mutants, indicating the existence of another pathway for producing H₂S in Z. mobilis. All these results would help to further elucidate the metabolic pathways involving H₂S in Z. mobilis and exploit the biotechnological applications of this industrially important bacterium.     

Genetics of three esterase loci in Anopheles stephensi liston

A survey of laboratory strains of Anopheles stephensi for nonspecific esterases by polyacrylamide gel electrophoresis revealed 10 zones of esterase activity. In 3 of the 10 zones, three electromorphs were observed. Genetic analysis revealed that these three zones are controlled by three loci, viz., Est-3, Est-4, and Est-5, and that the electromorphs are codominant alleles at each locus. The three esterase loci were found linked to each other and to an autosomal marker colorless-eye. The esterase loci have tentatively been placed in linkage group II. The probable gene sequence on chromosome 2 is either c-Est-3-Est-4-Est-5 or c-Est-4-Est-3-Est-5.     

Analysis of zebrafish Mhc using BAC clones

 Although major histocompatibility complex (Mhc) genes have been identified in a number of species, little is yet known about their organization in species other than human and mouse. The zebrafish, Danio rerio, is a good candidate for full elucidation of the organization of its Mhc. As a step toward achieving this goal, a commercially available zebrafish BAC library was screened with probes specific for previously identified zebrafish class I and class II genes, as well as for genes controlling the proteasome subunits LMP7 and LMP2. Restriction maps of the individual positive clones were prepared and the Mhc (LMP7) genes localized to specific fragments. The total length of genomic DNA fragments with Mhc genes was approximately 1700 kilobases (kb) (200 kb of fragments bearing class I loci and 1500 kb of fragments bearing class II loci). One of the two class I loci (Dare-UCA) is closely associated with the LMP7 locus; the second class I locus (Dare-UAA) is more than 50 kb distant from the UCA locus and has no LMP genes associated with it. None of the class II genes are linked to the class I or the LMP genes. All six of the previously identified class II B genes and one of the three class II A genes were found to be present in the BAC clones; no new Mhc loci could be identified in the library. Each of the six previously identified class II B loci was found to be borne by a separate group of BAC clones. The Dare-DAB and -DAA loci were found on the same clone, approximately 15 kb apart from each other. An expansion of DCB and DDB loci was detected: the zebrafish genome may contain at least five closely related DCB and two closely related DDB loci which are presumably the products of relatively recent tandem duplication. These results are consistent with linkage studies and indicate that in the zebrafish, the class I and class II loci are on different chromosomes, and the class II loci are in three different regions, at least two of which are on different chromosomes. Received: 14 August 1997 / Revised: 16 September 1997