Products Of Structural Genes Research Articles

Comparative genomic analysis of five pairs of virulent parental/attenuated vaccine strains of PRRSV

Porcine reproductive and respiratory syndrome virus (PRRSV), the causative agent of porcine reproductive and respiratory syndrome, is responsible for serious disease in pigs resulting in substantial economic losses in the porcine industry. An attenuated vaccine strain, HuN4-F112, was obtained by passaging virulent PRRSV strain HuN4 on Marc-145 cells (for 112 passages), and the full-genomic sequence was determined. To understand the molecular basis of attenuation of PRRSV, we compared and analyzed the genomic sequences of HuN4/HuN4-F112, together with those of other four virulent parental/attenuated vaccine strains. Among the 19 PRRSV proteins, two (NSP6 and NSP8) were highly conserved, without any mutations and considered irrelative to attenuation. The mutation rates of envelope-associated structural proteins were obviously higher than those of most non-structural proteins. It is interesting that the gene of the smallest structural protein, E protein, had the highest mutation rate among all of the structural genes analyzed, and also harbored a highly variable region. Our results indicate that determinants of PRRSV attenuation are multigenic products of both non-structural and structural genes. To our knowledge, this is the first report showing that the envelope-associated structural proteins (including E and GP2–GP5 proteins) may play a significant role. These findings contribute towards our understanding of PRRSV attenuation and will provide an important clue for further study.

Bacterial gene products in response to near-ultraviolet radiation

Our research has focused on bacterial gene products that protect cells from damage by near-ultraviolet radiation (near-UV) including gene products involved in the subsequent recovery process. Protective gene products include such anti-oxidants as catalases, superoxide dismutases and glutathione reductase. Near-UV damage recovery products include exonuclease III and DNA-glycosylases. Perhaps more critical than the products of structural genes are certain regulatory gene products that are triggered upon excess near-UV oxidation and lead to synthesis of entire batteries of anti-oxidant enzymes, DNA repair enzymes, and DNA-integrity proteins. Our recent experiments have focused on RpoS and its interaction with OxyR, two proteins that regulate the synthesis of molecules that protect cells from near-UV and other oxidative stresses.

Purification and characterization of citrate synthase isoenzymes from Pseudomonas aeruginosa.

Two types of citrate synthase (CS) have been purified from Pseudomonas aeruginosa, a 'large' form (CSI) and a 'small' form (CSII). The M(r)s of the CSI and CSII isoenzymes were determined to be 240,000 +/- 16,000 (mean +/- S.E.M.) and 80,300 +/- 3800 respectively. Chemical cross-linking of the native enzymes with either dimethyl suberimidate or glutaraldehyde followed by electrophoretic analysis by SDS/PAGE showed that CSI is a hexamer and CSII is a dimer. SDS/PAGE showed that CSI and CSII each consist of a single subunit type, of M(r) 42,000 +/- 2000 and M(r) 36,500 +/- 2000 respectively. CSI and CSII were also shown to be distinct kinetically, immunologically and in terms of their regulatory properties. It is suggested that the CS isoenzymes are products of different structural genes.

Identification of a Serratia entomophila genetic locus encoding amber disease in New Zealand grass grub (Costelytra zealandica).

Serratia entomophila UC9 (A1MO2), which causes amber disease in the New Zealand grass grub Costelytra zealandica, was subjected to transposon (TnphoA)-induced mutagenesis. A mutant (UC21) was found to be nonpathogenic (Path-) to grass grub larvae in bioassays and was shown, by Southern hybridization, to contain a single TnphoA insertion. This mutant failed to adhere to the gut wall (Adn-) of the larvae and also failed to produce pili (Pil-). A comparative study of the total protein profiles of wild-type S. entomophila UC9 and mutant UC21 revealed that the mutant lacked an approximately 44-kDa protein and overexpressed an approximately 20-kDa protein. Transfer of cosmids containing homologous wild-type sequences into mutant strain UC21 restored wild-type phenotypes (Path+, Pil+, and Adn+). One of the complementing cosmids (pSER107) conferred piliation on Pil- Escherichia coli HB101. The TnphoA insertion in UC21 was mapped within an 8.6-kb BamHI fragment common to the complementing cosmids, and we designated this gene locus amb-1. Six gene products with molecular masses of 44, 36, 34, 33, 20, and 18 kDa were detected in E. coli minicells exclusive to the cloned 8.6-kb fragment (pSER201A). The 44-kDa gene product was not detected in E. coli minicells containing the cloned mutant fragment. Saturation mutagenesis of this fragment produced four unlinked insertional mutations with active fusions to TnphoA. These active fusions disrupted the expression of one or more gene products encoded by amb-1. The 8.6-kb fragment cloned in the opposite orientation (pSER201B) expressed only a 20-kDa protein. We propose that these are the products of structural and/or regulatory genes involved in adhesion and/or piliation which are prerequisites in the S. entomophila-grass grub interaction leading to amber disease.

Linkage relationships of loci controlling protein amounts in maritime pine (Pinus pinaster Ait.)

The comparison of two-dimensional protein patterns of 56 haploid megagametophytes from a single maritime pine tree allowed the analysis of genetic determinism of qualitative and quantitative variation. One hundred and nineteen loci were localized in 12 linkage groups while two other loci were unlinked. Among the 81 qualitative variants, 58 could be interpreted as allelic products of structural genes. Their distribution in the linkage groups appeared to be non-random, since most of them clustered at a common point in two groups. The remaining 23 qualitative and 40 quantitative variants could best be explained by assuming monogenic determinism of protein amounts. Pleiotropic effects could be responsible for some of the variations in amounts of proteins which cosegregate.

GENE PRODUCTS OF BARLEY CHROMOSOMES 4 AND 7 ARE PRECURSORS OF THE MAJOR ANTIGENIC BEER PROTEIN

Proteins in grains of wheat-barley chromosome addition lines and in a protein-enriched concentrate from beer (Fraction X) were compared by tandem crossed immunoelectrophoresis. Two major beer proteins, called Antigen 1a and 1b, were found to be products of distinct structural genes positioned on barley chromosomes 4 and 7, respectively.

Antibodies to Murine Amelogenins: Localization of Enamel Proteins During Tooth Organ Development in vitro

During mouse tooth development the secretory ameloblasts differentiate and produce a population of acidic glycoproteins termed ‘amelogenins’ which function in the formation of the extracellular enamel organic matrix. Amelogenin-enriched fractions were isolated from murine molar and incisor tooth organs during early postnatal enamel matrix production using an extraction procedure with acetic acid. Major polypeptides were separated by gel electrophoresis, amelogenin polypeptide bands (circa 16–20,000 molecular weight) were exicised, amelogenins were eluted from the gel bands and then used as immunogen for immunization. Rabbit antibodies raised against amelogenins showed extensive cross-reactivity with lower molecular weight polypeptides of 16,000–20,000. These antibodies were used with indirect immunofluorescence microscopy to show the specific localization of amelogenins during the synthesis and secretion of the enamel matrix in situ as well as in vitro. No staining reaction was found in keratinized or non-keratinized epidermal tissues, dental papilla mesenchyme, odontoblasts, dentine matrix, cartilage, bone, heart, brain, lung, or salivary glands. In order to detect differentiation-specific synthesis of amelogenins, we cultured early cap stage molar tooth organs (17-days gestation or Theiler stage 25) in a serumless, chemically-defined medium for periods up to 21 days. The anti-amelogenin antibodies did not stain, by indirect immunofluorescence, cap stage molar tooth organ tissues. However, within six days in vitro, explants stained with antisera and showed localization within ameloblasts and newly secreted enamel matrix. Comparisons with in situ epithelial differentiation into ameloblasts suggested that cultured explants required approximately twice as long to reach a comparable expression of amelogenins. Synthesis of amelogenins was first detected by immunoprecipitation also during the sixth day of organ culture. The family of amelogenin polypeptides may be products of structural genes which can become activated during epithelial-mesenchymal interactions, even in the absence of exogenous growth factors such as serum or neutrotrophic mediators. The expression of amelogenins, therefore, does not appear to require hormones and may be an example of mesen-chyme-directed epithelial differentiation.

Avian scale development: VIII. Relationships between morphogenetic and biosynthetic differentiation

Epidermal-dermal tissue interactions during development control not only the morphogenesis of skin derivatives, but also the synthesis of the α- and β-keratins, which are the products of distinct structural genes. The electrophoretic patterns of the s-carboxymethyl (SCM) derivatives of the keratins produced by either embryonic or hatched chick epidermis from normal overlapping scales (scutate and scutellate scales), the radially symmetrical scales (reticulate scales), and the general body integument have been compared with the patterns from corresponding regions of birds homozygous for the gene, scaleless ( sc sc ). We find six polypeptides in the molecular weight range of α-keratin, three β-keratins, and one major polypeptide of an intermediate molecular weight; any of which may or may not be present in a given tissue. Our comparisons show (1) that for some regions of the integument there are inconsistencies between the data obtained from our electrophoretic studies and those obtained from fine structural and X-ray diffraction studies (i.e., some regions which are fine structurally distinct give identical SDS-PAGE patterns); (2) that the biosynthetic differentiation of the overlapping scales is dependent upon specific morphogenetic events, while the biosynthetic differentiation of the reticulate scales is not; and (3) that there is region-specific gene expression for the epidermal keratins of all scaled regions when compared with the keratins of all nonscaled regions of the integument.

Evidence that three structural genes code for human alkaline phosphatases.

The number of structural gene loci that code for the different molecular forms of human alkaline phosphatase is unknown. Physical properties of the enzymes, immunological data, chemical inhibition and genetic studies suggest that at least three structural genes are involved: one coding for alkaline phosphatase from placenta, another for the enzyme from intestine, and one or more for the enzymes from liver, kidney and bone. Badger and Sussman have shown that alkaline phosphatases from human liver and placenta are products of different structural genes, and Greene and Sussman have shown that alkaline phosphatase from a metastasised bronchogenic carcinoma was nearly identical to the enzyme from placenta. However, other tumour-associated alkaline phosphatases and the enzymes from normal tissue other than placenta and liver have not been identified by conclusive structural criteria, and thus it is not known whether these onco-alkaline phosphatases represent ectopic production or unusual post-translational modification of the enzymes found in normal tissues. We present here, using a sensitive peptide-mapping technique, structural evidence that the enzyme forms from liver, kidney and serum from a patient with Paget's disease of bone (osteitis deformans) are products of the same structural gene and can be easily distinguished from either the intestinal or placental isoenzymes. The technqiue seems to be useful for the classification of tumour-associated alkaline phosphatases on a structural basis.

Radioimmune analysis of an unexpected mouse allotype.

We have used a radioimmune assay to confirm our earlier findings of an unexpected immunoglobulin allotype in Igb-congenic BALB/c mice. Although these mice were bred to exclude the IgG2a allotype of BALB/c (Ig-la), an Ig-la-like antigen was detected in the 7S Ig fraction of two (of five) pooled serum samples, it represented 0.1--0.3% of the total 7S protein and was indistinguishable from a reference Ig-la. The detection of putative Ig-la in Igb-congenic mice is inconsistent with the notion that allotypes are products of allelic structural genes. It appears rather that expression of Ig-la is controlled by allelic regulator genes and that its low and transient production in Igb-congenic mice results from incomplete negative regulation.

Characterization of Major Outer Membrane Proteins O-8 and O-9 of Escherichia coli K-12

Outer membrane proteins O-8 and O-9 have been highly purified from a strain of Escherichia coli K-12 by Sephadex G-200 and DEAE-cellulose chromatographies. The amino acid compositions of the purified proteins were definitely different, although they showed marked similarities. The profiles of BrCN peptides of the two proteins were also different. None of the BrCN peptides were the same for the two proteins. Analysis of the first twelve N-terminal residues revealed that the two proteins are strikingly similar, but with differences in the third and the eleventh amino acid residues. It can be concluded that proteins O-8 and O-9 are products of different structural genes which developed by duplication of an ancestral genome followed by mutation.

The identification of mutants ofEscherichia colideficient in formate dehydrogenase and nitrate reductase activities using dye indicator plates

The anaerobic, proton translocating, formatedependent nitrate reductase activity of Escherichia coli has been studied extensively, particularly with respect to the identification of the various redox components required, their functional organization in the cytoplasmic membrane, and the structural genes responsible for their synthesis [1,2]. The information so far available is summarized in Fig. 1 from which it is apparent that several outstanding problems remain. We have begun a detailed analysis of the molecular genetics of the system with particular emphasis on firstly the unambiguous characterizations of the products of the various structural genes so far described, and secondly the identification of the regulatory genes responsible for the control of structural gene expression. In this study it became apparent that many of the previously described methods for the identification and genetic characterization of relevant mutants were either time-consuming or imprecise. We describe here the application of two methods utilizing modified dye indicator plates which facilitate the rapid and unambiguous classification of mutants into those which: (i) specifically lack formate dehydrogenase activity; (ii) specifically lack D-lactate-dependent nitrate reductase activity; and (iii) pleiotropically lack both activities.

Genic heterozygosity and rate of speciation

The hypothesis is proposed that mean level of heterozygosity is functionally related to rate of speciation in evolutionary phylads. Under this hypothesis, phylads which speciate more rapidly do so because of increased level of within-species genetic variability which is then available to conversion to species differences under appropriate ecological or environmental conditions. An important corollary is that rate of speciation could be limited in phylads with low genetic variability, irrespective of environmental considerations.This hypothesis has been tested with respect to electrophoretically detectable variation in products of structural genes in two families of North American fishes characterized by grossly different rates of speciation. Totals of 69 species of the highly speciose Cyprinidae, and 19 species of the relatively depauperate Centrarchidae, were assayed for mean level of heterozygosity at 11–24 genetic loci. Since Cyprinidae and Centrarchidae exhibit on the average nearly identical levels of genic variation (Ĥ= 0.052 ± 0.004, andĤ= 0.049 ± 0.009, respectively), the hypothesis that level of heterozygosity affects rate of speciation in these fishes is not supported.Nonetheless, the amount of genic variability in both Cyprinidae and Centrarchidae is large, comparable to mean levels in previously studied vertebrates. The great wealth of genome variability, reflected in the electrophoretic variation present in virtually all outcrossing organisms, apparently can accommodate considerable flexibility in rate and pattern of evolutionary response to the various environmental regimes challenging organisms.

Anomaly in the gamma chain heterogeneity of the newborn.

THE two types of γ chain in human foetal haemoglobin (HbF) are the products of nonallelic structural genes and have either glycine (Gγ type) or alanine (Aγ type) in position 136 (ref. 1). Although the examination of 108 cord blood samples from worldwide sources showed 102 to have a Gγ : Aγ ratio within a relatively narrow range, one had a low ratio and five a high ratio2. It seemed possible that analytical error was responsible for these extreme values. But as we studied cord blood from normal black babies of residents from Burke County, Georgia, where an abnormal HbF (designated FX (ref. 3)) has been detected repeatedly, we observed high ratios in further samples. The present study of almost 700 cord bloods has detected the high Gγ : Aγ ratio in about 7.5% of samples from black newborns as compared with perhaps 0.5% from Caucasian infants.

Chick cytoplasmic actin and muscle actin have different structural genes.

Actins isolated from embryonic chick brain and muscle differ in mobility when subjected to electrophoresis in gels containing urea and sodium dodecyl sulfate. Experiments were carried out to determine whether these actins are products of different structural genes and differ in primary amino acid sequence, or whether they are products of the same structural gene but are different because of post-translational modification. Messenger RNA from brain and muscle tissue was used to direct cell-free protein synthesis in wheat germ extracts. The synthesized actins were identified by conversion from globular to fibrous actin and by two-dimensional chromatographic analysis of tryptic peptides. The differences in electrophoretic mobility of brain compared to muscle actin were maintained in the cell-free protein synthetic products. Therefore, these mobility differences were not due to post-translational modification. It was concluded that brain and muscle actin are coded by different messenger RNAs and therefore arise from different structural genes. In addition, messenger RNA from 13- and 16-day embryonic thigh muscle directed the synthesis of both brain- and muscle-type actins, suggesting that muscle cell differentiation involves the regulation of at least two different actin genes.

Beta-ketoadipate enol-lactone hydrolases I and II from Acinetobacter calcoaceticus

Beta-Ketoadipate enol-lactone hydrolase catalyzes a common step in the utilization of protocatechuate and cis,cis-muconate by bacteria. Either of the two compounds elicits the synthesize of an enol-lactone hydrolase in Acinetobacter. The enol-lactone hydrolase that is induced by each compound was purified, and the properties of the proteins were compared. Both enzymes appear to be dimers with molecular weights of approximately 25,000. The amino acid compositions of the enzymes differ, and the two proteins do not cross-react serologically. The NH2-terminal amino acid residue of the protocatechuate-induced enol-lactone hydrolase (ELH I) is methionine and the NH2-terminal amino acid residue of the cis,cis-muconate-induced enol-lactone hydrolase (ELH II) is proline. Therefore, ELH I and ELH II appear to be the products of different structural genes. The serological specificity of ELH I and ELH II made it possible to demonstrate the mutually independent regulation of their synthesis in wild type cells and in constitutive mutant strains. The synthesis of ELH I is not impaired in mutant strains that cannot synthesize ELH II. The rapid characterization of mutant strains that produce ELH I or ELH II constitutively was made possible by the development of pH indicator enzyme assays that were performed with toluenized cells. cis,trans-Muconate, which does not support the growth of Acinetobacter, elicits the synthesis of the enzymes that normally are induced by cis,cis-muconate to 20% of fully induced levels.

Rabbit Immunoglobulin Allotypes: Structure, Immunology, and Genetics

This chapter presents the allotypic studies in the rabbit and offers more detailed information on several specific topics to which allotypic studies have contributed. The chapter focuses on the rabbit allotypes and gives an overview of the developments. There is a paucity of structural information on rabbit immunoglobulins and an optimal amount of genetic information has been obtained in studies on this species by the use of the immunoglobulin antigenic markers. The chapter explains allotypic phenomena adequately by involving regulator genes in the linkage, somatic recombination, and selective gene expression that have been observed in genetic studies employing allotypes. It is difficult to visualize these complex genetic relationships in terms of the repressors and corepressors that have been described for nonmammalian systems involving regulation. The possibility has been raised that rabbit immunoglobulin allotypes are not products of allelic structural genes, however are products of regulator genes. The studies on allotypes and idiotypes have made significant contributions to the knowledge of genetic control of immunoglobulin synthesis, knowledge that is, in many instances, relevant to more general biological topics.

Metabolic role, regulation of synthesis, cellular localization, and genetic control of the glyoxylate cycle enzymes in Neurospora crassa.

The glyoxylate shunt enzymes, isocitrate lyase and malate synthase, were present at high levels in mycelium grown on acetate as sole source of carbon, compared with mycelium grown on sucrose medium. The glyoxylate shunt activities were also elevated in mycelium grown on glutamate or Casamino Acids as sole source of carbon, and in amino acid-requiring auxotrophic mutants grown in sucrose medium containing limiting amounts of their required amino acid. Under conditions of enhanced catabolite repression in mutants grown in sucrose medium but starved of Krebs cycle intermediates, isocitrate lyase and malate synthase levels were derepressed compared with the levels in wild type grown on sucrose medium. This derepression did not occur in related mutants in which Krebs cycle intermediates were limiting growth but catabolite repression was not enhanced. No Krebs cycle intermediate tested produced an efficient repression of isocitrate lyase activity in acetate medium. Of the two forms of isocitrate lyase in Neurospora, isocitrate lyase-1 constituted over 80% of the isocitrate lyase activity in acetate-grown wild type and also in each of the cases already outlined in which the glyoxylate shunt activities were elevated on sucrose medium. On the basis of these results, it is concluded that the synthesis of isocitrate lyase-1 and malate synthase in Neurospora is regulated by a glycolytic intermediate or derivative. Our data suggest that isocitrate lyase-1 and isocitrate lyase-2 are the products of different structural genes. The metabolic roles of the two forms of isocitrate lyase and of the glyoxylate cycle are discussed on the basis of their metabolic control and intracellular localization.

Implications of heavy chain disease protein for multiple gene theories of immunoglobulin synthesis

The sequence of the amino-terminal 34 amino acids of 'y-heavy chain disease (y-HCD) protein Hi is homologous with the amino-terminal region of immunoglobulin heavy chains. 7-heavy chain disease is smaller than a normal y-chain, but has the carboxy-terminal composition expected for 7y-chains and must, therefore, contain an internal deletion. Comparison of the Hi sequence with that of y-heavy chain disease Zu, which also has an internal deletion, indicates that the site of internal deletion is not a constant characteristic of 7-heavy chain disease proteins. Heavy chains can be assigned to subgroups on the basis of variable region sequences. The variable regions of Hi and one other protein differ significantly from those determined for other heavy chains, and these two proteins are assigned to a new heavy chain variable region subgroup, VHIV. It has been suggested that single immunoglobulin heavy chains are the products of two separate structural genes and that variable region genetic information is translocated and integrated into common region information. These multiple gene theories make no prediction as to whether DNA or RNA is translocated. 7y-heavy chain disease proteins provide unique information that indicates that if translocation is required for the production of immunoglobulin heavy chains, it is DNA, not RNA, that is translocated. Introduction. Heavy chain disease (HCD) proteins are aberrant forms of human immunoglobulin heavy polypeptide chains. Examples of heavy chain disease proteins have been described for the classes IgG,1-7 IgA,8'9 and IgM.10 Studies of IgG HCD (y-HCD) proteins have shown that they consist of portions of the y-chain and that they lack light polypeptide chains. The two 7y-HCD proteins for which data are available have molecular weights of 26,00011 and 40,0007 as compared with a molecular weight of approximately 54,000 for normal 7-chains.12 The structure of y-HCD proteins is of interest because, at least in some cases, they appear to be the intact products of mutated immunoglobulin structural genes, rather than the partially degraded products of normal immunoglobulin structural genes. It was first shown by Prahl3 that the amino acid sequence of the amino-terminal (N-terminal) tripeptide of 7-HCD Zu was similar to the sequence determined for the amino-terminus of 7-chains.'4 Moreover, it was

The heterogeneity of the fetal hemoglobin of the goat

The heterogeneity of the goat fetal hemoglobin, which has been observed by measurements of the rate of denaturation in an alkaline medium, was reinvestigated. Analyses of the amino acid compositions of various peptides isolated from a tryptic digest of the γ chain failed to demonstrate a molecular heterogeneity. Alkali-induced denaturation rates of red cell hemolysates from normal newborn goats, and from newborn goats with a heterozygosity or homozygosity for the Hb I αB allele, indicated the presence of two distinct components in each of these hemolysates. Similar experiments with isolated fetal hemoglobins containing either I α B or II α polypeptide chains gave distinctly different denaturation curves, which obeyed first order kinetics. It was concluded that the presence of two structurally different α chains, which are the products of non-allelic structural genes, is responsible for the observed phenomenon; the differences in rate of alkali-induced denaturation apparently result from differences in the stability of the II αγ and I αγ dimer subunits.