Gene expression in the hippocampus of inbred alcohol-preferring and -nonpreferring rats

The hippocampus is sensitive to the effects of ethanol and appears to have a role in the development of alcohol tolerance. The objective of this study was to test the hypothesis that there are innate differences in gene expression in the hippocampus of inbred alcohol-preferring (iP) and -nonpreferring (iNP) rats that may contribute to differences in sensitivity to ethanol and/or in the development of tolerance. Affymetrix microarrays were used to measure gene expression in the hippocampus of alcohol-naive male iP and iNP rats in two experiments (n=4 and 6 per strain in the two experiments). Combining data from the two experiments, there were 137 probesets representing 129 genes that significantly differed (P < or = 0.01); 62 probesets differed at P < or = 0.001. Among the 36% of the genes that were expressed more in the iP than iNP rat at this level of significance, many were involved in cell growth and adhesion, cellular stress reduction and anti-oxidation, protein trafficking, regulation of gene expression, synaptic function and metabolism. Among the 64% of the genes that had lower expression in the hippocampus of iP than iNP rats were genes involved in metabolic pathways, cellular signaling systems, protein trafficking, cell death and neurotransmission. Overall, the data indicate that there are significant innate differences in gene expression in the hippocampus between iP and iNP rats, some of which might contribute to the differences observed in the development of alcohol tolerance between the selectively bred P and NP lines.     

Innate differences in protein expression in the nucleus accumbens and hippocampus of inbred alcohol-preferring and -nonpreferring rats

Two-dimensional gel electrophoresis (2-DE) was used to separate protein samples solubilized from the nucleus accumbens and hippocampus of alcohol-na?ve, adult, male inbred alcohol-preferring (iP) and alcohol-nonpreferring (iNP) rats. Several protein spots were excised from the gel, destained, digested with trypsin, and analyzed by mass spectrometry. In the hippocampus, 1629 protein spots were matched to the reference pattern, and in the nucleus accumbens, 1390 protein spots were matched. Approximately 70 proteins were identified in both regions. In the hippocampus, only 8 of the 1629 matched protein spots differed in abundance between the iP and iNP rats. In the nucleus accumbens, 32 of the 1390 matched protein spots differed in abundance between the iP and iNP rats. In the hippocampus, the abundances of all 8 proteins were higher in the iNP than iP rat. In the nucleus accumbens, the abundances of 31 of 32 proteins were higher in the iNP than iP rat. In the hippocampus, only 2 of the 8 proteins that differed could be identified, whereas in the nucleus accumbens 21 of the 32 proteins that differed were identified. Higher abundances of cellular retinoic acid-binding protein 1 and a calmodulin-dependent protein kinase (both of which are involved in cellular signaling pathways) were found in both regions of the iNP than iP rat. In the nucleus accumbens, additional differences in the abundances of proteins involved in (i) metabolism (e.g., calpain, parkin, glucokinase, apolipoprotein E, sorbitol dehydrogenase), (ii) cyto-skeletal and intracellular protein transport (e.g., beta-actin), (iii) molecular chaperoning (e.g., grp 78, hsc70, hsc 60, grp75, prohibitin), (iv) cellular signaling pathways (e.g., protein kinase C-binding protein), (v) synaptic function (e.g., complexin I, gamma-enolase, syndapin IIbb), (vi) reduction of oxidative stress (thioredoxin peroxidase), and (vii) growth and differentiation (hippocampal cholinergic neurostimulating peptide) were found. The results of this study indicate that selective breeding for disparate alcohol drinking behaviors produced innate alterations in the expression of several proteins that could influence neuronal function within the nucleus accumbens and hippocampus.     

Changes in liver protein abundance in inbred alcohol-preferring rats due to chronic alcohol exposure, as measured through a proteomics approach

This study compares the total liver proteome of inbred alcohol‐preferring line (iP) rats exposed to alcohol with iP rats without alcohol experience. Rat liver proteins were extracted using a three‐step procedure. Each of the three solutions solubilizes a different set of proteins. The extracted proteins were separated by 2‐DE. Scanned gels of two sample groups, alcohol‐exposed iP and alcohol‐naïve iP, were compared, revealing many protein spots with significantly higher or lower densities. These spots were cut from the gel, destained, and subjected to trypsin digestion and subsequent identification by LC‐MS/MS. Twenty‐four individual rats, 12 alcohol‐naïve, and 12 alcohol‐exposed, were used in this study. Two groups, each containing six naïve and six exposed animals, were created for statistical comparison. For the first group, 64 spots were observed to have statistically significant intensity differences upon alcohol exposure across all three extracts while 118 such spots were found in the second group. There were 113 unique proteins in both groups together. The majority of these proteins were enzymes. Significant changes are observed for three major metabolic pathways: glycolysis, gluconeogenesis, and fatty acid β‐oxidation. In addition, enzymes involved in protein synthesis and antioxidant activity show significant changes in abundance in response to alcohol exposure.     

Phenylalanine Ammonia-Lyase Genes Involved in Anthocyanin Synthesis and the Regulation of its Expression in Suspension Cultured Carrot Cells

D and is also induced rapidly and transiently by transfer of cells to fresh medium and lowering the cell density. From the carrot genomic library, four clones of PAL genes, gDcPAL1,2,3 and 4, were obtained. Analyses of nucleotide sequences revealed that only the gDcPAL3 gene is responsible for the induction of anthocyanin synthesis by 2,4-D. Several cis-elements, boxes M, P, A, L, and G, exist in the proximal promoter region of gDcPAL3. Transient expression experiments in carrot protoplasts using deletion mutants of the proximal promoter region of gDcPAL3 gene showed that boxes M and L, both of which contain core sequences of the Myb binding sites, might play an important role in gDcPAL3 promoter activity. Four myb cDNAs, Dcmyb8,10,12 and 14 were obtained from a carrot subtracted cDNA library and their structure and expression patterns were analyzed. In addition to the analysis of the proximal region of gDcPAL3 promoter, the possibility of the regulation of gene expression by genomic DNA structure and chromatin modification in metabolic differentiation is discussed. Received 10 June 2000/ Accepted in revised form 1 July 2000     

Stress induction and developmental regulation of a rice chitinase promoter in transgenic tobacco

A 1.5 kb promoter fragment from the rice (Oryza sativa L.) RCH10 gene, which encodes a basic endochitinase inducible by wounding and fungal elicitor, was translationally fused to the β-glucuronidase (GUS) reporter gene and transferred to tobacco by Agrobacterium tumefaciens-mediated leaf disc transformation. Wounding of leaves induced GUS activity from low basal levels, and addition of fungal elicitor to the wounded tissue caused a further marked activation of the gene fusion. During vegetative development high levels of GUS activity were observed in roots and moderate levels in stems. Histochemical analysis indicated that the promoter was active in vascular and epidermal tissue, and the root apical tip. In flowers, high levels of GUS activity were observed in stigmas, ovaries and pollen-containing anthers, but only low levels in sepals and petals. The promoter 5′-deleted to ?160 exhibited the same patterns of expression in floral organs, and was also strongly induced by wounding and elicitor, but GUS activity was markedly reduced in vegetative organs. More detailed 5′ deletions showed that a cis-element required for floral expression was located between ?160 and ?74, and a cis element sufficient for stress induction was located 3′ of ?74. This proximal region 3′ of ?74 was also sufficient for expression in transfected rice protoplasts derived from suspension cultured cells. These data indicate that the complex developmental and environmental regulation of RCH10 promoter activity involves several distinct cis-elements for vegetative expression, floral expression and stress induction, and that signal pathways for wound and elicitor induction are conserved between monocotyledonous and dicotyledonous plants.     

Bicistronic expression strategy for high‐level expression of recombinant proteins in Corynebacterium glutamicum

Directly using the promoter associated with 5′‐untranslated region of a high‐protein‐abundance gene from the genome may cause low expression activity of an expression system. A bicistronic expression part containing the short 5′ coding sequence of the source gene and an embedded Shine–Dalgarno sequence can cause higher expression levels of the recombinant gene in a bicistronic cassette. Here, we evaluated two methods to construct expression parts and exploited genomic sequence sources to provide specific functional sequences to complete the expression system. The architecture of the bicistronic part increased the expression levels of target genes and performed more reliably than conventional expression parts with the same promoter and 5′ untranslated region. For Corynebacterium glutamicum, the strongest bicistronic part, HP‐BEP4, was obtained from a heterologous sequence source, leading to a 2.24‐fold increase in the expression level of fluorescent protein over constitutively expressed pXMJ19 or the production of more than 100 mg/L single‐chain variable fragment (scFv). It could meet the needs of overexpressing key genes in C. glutamicum.