Modulation of Gene Expression Made Easy

A new approach for modulating gene expression, based on randomization of promoter (spacer) sequences, was developed. The method was applied to chromosomal genes in Lactococcus lactis and shown to generate libraries of clones with broad ranges of expression levels of target genes. In one example, overexpression was achieved by introducing an additional gene copy into a phage attachment site on the chromosome. This resulted in a series of strains with phosphofructokinase activities from 1.4 to 11 times the wild-type activity level. In this example, the pfk gene was cloned upstream of a gusA gene encoding β-glucuronidase, resulting in an operon structure in which both genes are transcribed from a common promoter. We show that there is a linear correlation between the expressions of the two genes, which facilitates screening for mutants with suitable enzyme activities. In a second example, we show that the method can be applied to modulating the expression of native genes on the chromosome. We constructed a series of strains in which the expression of the las operon, containing the genes pfk, pyk, and ldh, was modulated by integrating a truncated copy of the pfk gene. Importantly, the modulation affected the activities of all three enzymes to the same extent, and enzyme activities ranging from 0.5 to 3.5 times the wild-type level were obtained.     

Characterization of 3-Iodothyronamine In Vitro Dynamics by Mathematical Modeling

3-Iodothyronamine (T₁AM) is regarded as a hormone-like substance thanks to its endogenous nature, its interaction with specific receptors trace amine-associated receptor 1 and its biological effects. We characterized T₁AM transport and conversion in an in vitro culture of H9c2 murine cells, after a T₁AM bolus injection. Samples of cell medium culture and cell lysate were assayed by high-performance liquid chromatography coupled to tandem mass spectrometry. We performed comparative experiments by adding to T₁AM bolus amino oxidase inhibitors as iproniazid, pargyline (monoamine oxidase, MAO inhibitors), aminoguanidine, and semicarbazide (semicarbazide-sensitive amino oxidase, SSAO inhibitors). A mathematical model was developed, based on the assumption that T₁AM is transported with a mechanism that is typical of hormone transport (i.e., EGF or insulin). We noticed that surface receptors downregulation could play a major role in T₁AM dynamics. We also estimated that T₁AM catabolism is mainly affected by MAO inhibitors, which produce a dramatic decrease in the kinetic constants related to T₁AM degradation, while no significant changes were observed in experiments with SSAO inhibitors.     

Genetic Architecture of Physiological Phenotypes: Empirical Evidence for Coadapted Gene Complexes

Physiological phenotypes are the result of the coordinated functionof many genes, some of which may be differentiated between conspecificpopulations. Within any one population, natural selection willfavor evolution of a coadapted set of alleles which optimizesphysiological performance and reproductive success. The existenceof such coadapted gene complexes may be assessed by assayingphenotypes of interpopulation hybrids: inferior performanceof hybrids suggests that the allelic combinations present inthe parental populations are coadapted. This approach has beenused to examine the genetic architecture of physiological traitsin the copepod Tigriopus californicus, a species characterizedby sharp genetic differentiation of populations. Developmentaltime and response to osmotic stress both show pronounced F₂hybrid breakdown, a result consistent with genetic coadaptationwithin populations. To better understand the biochemical andmolecular mechanisms underlying hybrid breakdown, we are investigatinga specific biochemical phenotype, the activity of the enzymecytochrome c oxidase (COX). COX (encoded by multiple nuclearand mitochondrial genes) catalyzes the oxidation of cytochromec (encoded by a nuclear gene). Two approaches are being usedto address the extent of coadaptation (both among nuclear genesand between nuclear and mitochondrial genes) underlying COXfunction: (1) studies of the DNA (and inferred amino acid) sequencesof component genes among populations in search of coordinatepatterns of amino acid substitution across loci, and (2) directstudies of COX function in interpopulation hybrids and backcrosses.These approaches provide evidence for the existence of nuclear/nuclearand/or nuclear/mitochondrial coadaptation within natural populationsof T. californicus.     

A Similar Dichotomy of Sugar Modulation and Developmental Expression Affects Both Paths of Sucrose Metabolism: Evidence from a Maize Invertase Gene Family

Invertase and sucrose synthase catalyze the two known paths for the first step in carbon use by sucrose-importing plant cells. The hypothesis that sugar-modulated expression of these genes could provide a means of import adjustment was initially suggested based on data from sucrose synthases alone; however, this hypothesis remained largely conjectural without critical evidence for invertases. Toward this end, a family of maize invertases was cloned and characterized. Here, we show that invertases are indeed sugar modulated and, surprisingly, like the sucrose synthase genes, fall into two classes with contrasting sugar responses. In both families, one class of genes is upregulated by increasing carbohydrate supply (Sucrose synthase1 [Sus1] and Invertase2 [Ivr2]), whereas a second class in the same family is repressed by sugars and upregulated by depletion of this resource (Shrunken1 [Sh1] and Invertase1 [Ivr1]). The two classes also display differential expression during development, with sugar-enhanced genes (Sus1 and Ivr2) expressed in many importing organs and sugar-repressed, starvation-tolerant genes (Sh1 and Ivr1) upregulated primarily during reproductive development. Both the Ivr1 and Ivr2 invertase mRNAs are abundant in root tips, very young kernels, silk, anthers, and pollen, where a close relationship is evident between changes in message abundance and soluble invertase activity. During development, patterns of expression shift as assimilate partitioning changes from elongating silks to newly fertilized kernels. Together, the data support a model for integrating expression of genes differentially responsive to carbohydrate availability (i.e., feast and famine conditions) with developmental signals. The demonstration that similar regulatory patterns occur in both paths of sucrose metabolism indicates a potential to influence profoundly the adjustment of carbon resource allocation.     

Expression of the Arginine Regulon of Escherichia coli W: Evidence for a Second Regulatory Gene

Effect of the M (modifier) gene of Escherichia coli W on the expression of wild-type structural genes of four arginine biosynthetic enzymes was studied by examining enzyme activity in cell-free extracts of cultures grown in minimal medium and medium containing arginine. The mutant M gene was originally identified as causing arginine-induced synthesis of acetylornithine delta-transaminase in a strain deficient for the enzyme. The strains used in this study received the mutant M gene by recombination. Noncoordinate repression has been demonstrated for two more enzymes of the arginine regulon of E. coli W and the M(-) gene increases the degree of noncoordinate repression for the regulon. Mutation of the M gene results in altered regulation of acetylornithine delta-transaminase, ornithine transcarbamylase, and acetylornithinase. In addition, a decreased growth rate is observed. It is proposed that the M gene is a regulatory gene. A model is presented to explain the data which involves changes in operator-repressor affinity for the structural genes and possibly for the gene controlling arginyl transfer ribonucleic acid synthetase.     

Modulation of Antimicrobial Host Defense Peptide Gene Expression by Free Fatty Acids

Routine use of antibiotics at subtherapeutic levels in animal feed drives the emergence of antimicrobial resistance. Development of antibiotic-alternative approaches to disease control and prevention for food animals is imperatively needed. Previously, we showed that butyrate, a major species of short-chain fatty acids (SCFAs) fermented from undigested fiber by intestinal microflora, is a potent inducer of endogenous antimicrobial host defense peptide (HDP) genes in the chicken (PLoS One 2011, 6: e27225). In the present study, we further revealed that, in chicken HD11 macrophages and primary monocytes, induction of HDPs is largely in an inverse correlation with the aliphatic hydrocarbon chain length of free fatty acids, with SCFAs being the most potent, medium-chain fatty acids moderate and long-chain fatty acids marginal. Additionally, three SCFAs, namely acetate, propionate, and butyrate, exerted a strong synergy in augmenting HDP gene expression in chicken cells. Consistently, supplementation of chickens with a combination of three SCFAs in water resulted in a further reduction of Salmonella enteritidis in the cecum as compared to feeding of individual SCFAs. More importantly, free fatty acids enhanced HDP gene expression without triggering proinflammatory interleukin-1β production. Taken together, oral supplementation of SCFAs is capable of boosting host immunity and disease resistance, with potential for infectious disease control and prevention in animal agriculture without relying on antibiotics.     

Evidence of New Risk Genetic Factor to Systemic Lupus Erythematosus: The UBASH3A Gene

The ubiquitin associated and Src-homology 3 (SH3) domain containing A (UBASH3a) is a suppressor of T-cell receptor signaling, underscoring antigen presentation to T-cells as a critical shared mechanism of diseases pathogenesis. The aim of the present study was to determine whether the UBASH3a gene influence the susceptibility to systemic lupus erythematosus (SLE) in Caucasian populations. We evaluated five UBASH3a polymorphisms (rs2277798, rs2277800, rs9976767, rs13048049 and rs17114930), using TaqMan® allelic discrimination assays, in a discovery cohort that included 906 SLE patients and 1165 healthy controls from Spain. The SNPs that exhibit statistical significance difference were evaluated in a German replication cohort of 360 SLE patients and 379 healthy controls. The case-control analysis in the Spanish population showed a significant association between the rs9976767 and SLE (Pc = 9.9E-03 OR = 1.21 95%CI = 1.07–1.37) and a trend of association for the rs2277798 analysis (P = 0.09 OR = 0.9 95%CI = 0.79–1.02). The replication in a German cohort and the meta-analysis confirmed that the rs9976767 (Pc = 0.02; Pc = 2.4E-04, for German cohort and meta-analysis, respectively) and rs2277798 (Pc = 0.013; Pc = 4.7E-03, for German cohort and meta-analysis, respectively) UBASH3a variants are susceptibility factors for SLE. Finally, a conditional regression analysis suggested that the most likely genetic variation responsible for the association was the rs9976767 polymorphism. Our results suggest that UBASH3a gene plays a role in the susceptibility to SLE. Moreover, our study indicates that UBASH3a can be considered as a common genetic factor in autoimmune diseases.     

Localization of a Novel X-Linked Progressive Cone Dystrophy Gene to Xq27: Evidence for Genetic Heterogeneity

Clinical reexamination and DNA linkage analysis were carried out in an X-linked progressive cone dystrophy (XLPCD) family, previously described by Pinckers and Timmerman in 1981. In a large pedigree segregating XLPCD, by use of > or = 27 markers spanning the entire X chromosome, a novel locus for XLPCD was identified in Xq27. All other regions on the chromosome could be excluded. Since this novel locus is distinct from previously identified genes or regions involved in XLPCD, we further establish genetic heterogeneity underlying this disease entity.     

洱海鲤属鱼类同域分化形成的分子遗传学证据

对洱海的 3种鲤鱼———洱海鲤 (C .barbatus)、春鲤 (C .longipectoralis)和杞麓鲤 (C .carpiochila)线粒体DNA(mtDNA)进行了限制性片段长度多态 (RFLP)分析和mtDNA控制区 (D loop)序列测定。RFLP分析结果表明 ,16种酶在洱海鲤、春鲤和杞麓鲤 3个种共 14个个体上 ,仅DraⅠ在春鲤的一个个体上检出一个位点的限制性片段长度多态 ,独立为单倍型Ⅱ ,3个种的其余 13个个体共享单倍型Ⅰ ,种间遗传距离为 0 %～ 0 0 0 75 % ,此种间差异值比已报道的鱼类的种间差异甚至种内差异都低得多。D loop区的序列测定结果表明 ,长为 4 4 9bp的序列共显示有 7个变异位点 ,均为碱基替换 ,变异比例为 1 5 6 % ,在 14个鲤鱼个体中共检出 6种单倍型 ,种间的遗传距离为 0 0 82 %～ 0 171% ,与已研究的其他几种异域分布的鲤鱼相比 ,也至少小一个数量级。mtDNA的RFLP及D loop区的序列比较都揭示出洱海 3种鲤鱼之间的遗传差异远小于异域分布的鲤属鱼类物种之间及其他鱼类的同属种间的遗传差异。对洱海鲤属鱼类种间如此小的遗传差异唯一合理的解释就是 :它们确是由同域分化而形成的。     

Genetic Enhancement of Cold Tolerance by Expression of a Gene for Chloroplast [omega]-3 Fatty Acid Desaturase in Transgenic Tobacco

The increased production of trienoic fatty acids, hexadecatrienoic (16:3) and linolenic (18:3) acids, is a response connected with cold acclimation of higher plants and is thought to protect plant cells against cold damage. Transgenic tobacco (Nicotiana tabacum cv SR1) plants that contain increased levels of 16:3 and 18:3 fatty acids, and correspondingly decreased levels of their precursors, hexadecadienoic and linoleic acids, were engineered by introduction of a chloroplast [omega]-3 fatty acid desaturase gene (the fad7 gene) isolated from Arabidopsis thaliana. When exposed to 1[deg]C for 7 d and then cultured at 25[deg]C, the suppression of leaf growth observed in the wild-type plants was significantly alleviated in the transgenic plants with the fad7 gene. The low-temperature- induced chlorosis was also much reduced in the plants transformed with the fad7 gene. These results indicate that increased levels of trienoic fatty acids in genetically engineered plants enhance cold tolerance.     

Cloning, Tissue Expression Pattern, and Chromosome Location of a Novel Human Gene BRI3BP

A novel cDNA fragment was identified from a human fetal brain cDNA library by using the coding sequence of human BRI3 gene (Accession No. NM015379) as bait in a yeast two-hybrid screening. Then by 5'-RACE (rapid amplification of cDNA end) and electronic hybridization, we obtained a 1.9 kb contig which consists of a novel gene. It was designated as BRI3BP by the HUGO Nomenclature Committee. It contains an open reading frame encoding 251 amino acids. The calculated molecular weight of the deduced protein is 27.8 kU. The predicted isoelectric point is 9.48. Northern hybridization showed its mRNA was highly expressed in brain, kidney, and liver. By RH mapping, the BRI3BP gene was mapped to human chromosome 12q24.2-qter