Evolution of the expression of the Gld gene in the reproductive tract of Drosophila.

During the preadult development of Drosophila melanogaster, the GLD (glucose dehydrogenase) gene (Gld) is expressed in a variety of tissues, including the immature reproductive tract. At the adult stage the expression of Gld becomes largely restricted to the reproductive tract of males and females. We examined the expression of GLD in the adult reproductive tract of 50 species in the genus Drosophila, as well as in those of a few representative species from four other closely related genera. GLD exhibits considerable organ-specific diversity in the reproductive tract of males and females. Among these species, five male GLD phenotypes and six female GLD phenotypes were found. In contrast, the preadult expression of GLD in representative species from each distinct adult pattern type was determined and found to be highly conserved in both the immature reproductive tract and non-reproductive organs. Moreover, the set of reproductive organs that express GLD during preadult development is equivalent to the sum of the five male and six female adult GLD phenotypes. To initially define the contribution of cis- versus trans-acting factors responsible for differences in adult GLD expression between two of these species--D. melanogaster and D. pseudoobscura--we transferred the D. pseudoobscura Gld to the genome of D. melanogaster and investigated its expression. GLD expression patterns of these transformants displayed characteristics that are unique to both species, suggesting the presence of both cis- and trans-acting differences between these two species.     

Coevolution of the glucose dehydrogenase gene and the ejaculatory duct in the genus Drosophila

The glucose dehydrogenase gene (Gld) in Drosophila melanogaster exhibits a unique spatial and temporal pattern of expression. GLD expression switches from a non-sex-limited state at the pupal stage to a male-limited state at the adult stage. At the adult stage, the enzyme is restricted to the ejaculatory duct. Within the genus Drosophila, the ejaculatory duct has undergone a simple morphological divergence. In order to determine whether correlated changes in GLD expression had occurred, GLD activity during the pupal and adult stages was determined for several Drosophila species. It was found that virtually all of the species exhibit pupal GLD activity, whereas only those species with an expanded ejaculatory duct express male-limited GLD. The results of interspecific genital imaginal disc transplantation experiments indicate that the expanded morphology and GLD expression do not require any species- or sex-specific diffusible factors. An apparent regulatory polymorphism exists within the D. takahashii species with respect to male-limited GLD expression.      

Evolution of the glucose dehydrogenase gene in Drosophila

The glucose dehydrogenase genes (Gld) of Drosophila melanogaster, of D. pseudoobscura, and of D. virilis have been isolated and compared with each other in order to identify conserved and divergent aspects of their structure and expression. The exon/intron structure of Gld is conserved. The Gld mRNAs are similar, with a range of 2.6-2.8 kb among the three species. All three species exhibit peaks of Gld expression during every major developmental stage, although considerable variation in the precise timing of these peaks exists between species. Interspecific gene transfer experiments demonstrate that the regulation and function of the D. pseudoobscura Gld is similar enough to the homologous gene in D. melanogaster to substitute for its essential role in the eclosion process. Comparison of the putative promoter sequences has identified both shared and divergent sequence elements which are likely responsible, respectively, for the conserved and divergent patterns of expression observed. The entire coding sequences of the pseudoobscura and melanogaster Gld genes are presented and shown to encode a 612-amino-acid pre-protein. The inferred amino acid sequences are 92% conserved between the two species. In general the intronic regions of Gld are unusually well conserved.     

Complex organization of promoter and enhancer elements regulate the tissue- and developmental stage-specific expression of the Drosophila melanogaster Gld gene

The Drosophila melanogaster Gld gene has multiple and diverse developmental and physiological functions. We report herein that interactions among proximal promoter elements and a cluster of intronically located enhancers and silencers specify the complex regulation of Gld that underlies its diverse functions. Gld expression in nonreproductive tissues is largely determined by proximal promoter elements with the exception of the embryonic labium where Gld is activated by an enhancer within the first intron. A nuclear protein, GPAL, has been identified that binds the Gpal elements in the proximal promoter region. Regulation of Gld in the reproductive organs is particularly complex, involving interactions among the Gpal proximal promoter elements, a unique TATA box, three distinct enhancer types, and one or more silencer elements. The three somatic reproductive organ enhancers each activate expression in male and female pairs of reproductive organs. One of these pairs, the male ejaculatory duct and female oviduct, are known to be developmentally homologous. We report evidence that the other two pairs of organs are developmentally homologous as well. A comprehensive model to explain the full developmental regulation of Gld and its evolution is presented.     

Organ-specific patterns of gene expression in the reproductive tract of Drosophila are regulated by the sex-determination genes

The sex-determination genes of Drosophila act to repress the developmental pathway for the internal somatic reproductive organs of the opposite sex. By misregulating this pathway during preadult development, the organ-specific expression pattern of the glucose dehydrogenase gene (Gld) in the reproductive tract of adult flies has been changed without a concomitant sexual transformation of the reproductive organs. Misregulation of the tra, tra-2, and dsx genes leads to very similar patterns of ectopic expression of Gld. The induced ectopic patterns of Gld expression at the adult stage occur in a small subset of organs which all normally express the Gld gene during their morphogenesis. These ectopic patterns are irrevocably set during late larval-early pupal development. The normal pattern of Gld expression in several other Drosophila species is quite similar to the ectopic patterns which we have generated in D. melanogaster, suggesting that the interspecific variation in Gld expression may result from variation in the expression of the sex-determination genes.     

地高辛标记反意RNA探针检测脑组织切片生长抑素mRNA

本实验采用含大鼠生长抑素基因的ｐＳＰ６５ｃＤＮＡ质位通过转化至噬菌体内大量扩增,经提取,纯化后,用限制性内切酶进行酶切使质枝线性化,并将其作为模板,用地高辛（ＤｉｇｏｘｉｇｅｎｉｎＤｉｇ）作为标记物,体外转录合成生长抑素反意ＲＮＡ（ｃＲＮＡ）探针。实验动物选用ｗｉｓｔａｒ新生大鼠。冰冻切片,端、间脑切片经杂交前用Ｄｉｇ－ＵＴＰ标记的ｃＲＮＡ探针杂交,杂交后用抗Ｄｉｇ－碱性磷酸酶复合物进行酶联免疫反应。Ｘ－磷酸盐－ＮＢＴ显色。结果显示新生大鼠脑内生长抑素ｍＲＮＡ神经元着紫蓝色。杂交反应物集中于核周的胞浆及短小的突起内。胞核不着色。胞体轮廓清晰,周围背底浅淡。结果表明Ｄｉｇ标记ｃＲＮＡ探针不仅具备非同位素标记探针的优点而且能快速和准确检测组织细胞内ｍＲＮＡ的表达。     

Simple RNAi vectors for stable and transient suppression of gene function in rice

Since the recent sequencing of the rice genome, the functional identification of rice genes has become increasingly important. Various tagged lines have been generated; however, the number of tagged genes available is not sufficient for extensive study of gene function. To help identify the functions of genes in rice, we developed a Gateway vector, pANDA, for RNA interference of rice genes. This vector can be used for Agrobacterium transformation of rice and allows easy and fast construction of efficient RNAi vectors. In the construct, hairpin RNA derived from a given gene is transcribed from a strong maize ubiquitin promoter, and an intron is placed 5' upstream of inverted repeats to enhance RNA expression. Analysis of rice genes using this vector showed that suppression of mRNA expression was observed in more than 90% of transgenic plants examined, and short interfering RNA indicative of RNA silencing was detected in each silenced plant. A similar vector, pANDA-mini, was also developed for direct transfer into leaf cells or protoplasts. This vector can be used for transient suppression of gene function in rice. These vectors should help identify the functions of rice genes whose tagged mutants are not available at present and complement existing methods for functional genomics of rice.     

Developmental Expression of the Glucose Dehydrogenase Gene in Drosophila Melanogaster

The Gld gene of Drosophila melanogaster is transiently expressed during every stage of development. The temporal pattern of Gld expression is highly correlated with that of ecdysteroids. Exogeneous treatment of third instar larvae with 20-hydroxyecdysone induces the accumulation of Gld mRNA in the hypoderm and anterior spiracular gland cells. During metamorphosis Gld is expressed in a variety of tissues derived from the ectoderm. In the developing reproductive tract, Gld mRNA accumulates in the female spermathecae and oviduct and in the male ejaculatory duct and ejaculatory bulb. These four organs are derived from closely related cell lineages in the genital imaginal disc. Since the expression of Gld is not required for the development of these reproductive structures, this spatial pattern of expression is most likely a fortuitous consequence of a shared regulatory factor in this cell lineage. At the adult stage a high level of the Gld mRNA is only observed in the male ejaculatory duct.     

The YYRR box: a conserved dipyrimidine-dipurine sequence element in Drosophila and other eukaryotes.

We have discovered a novel DNA sequence element in Drosophila which is based upon a CTGA tandem repeat. This element has been named the YYRR box to emphasize its dipyrimidine-dipurine nature which is predicted to have unusual structural features. Southern hybridization analysis of genomic DNA indicates the presence of 25-30 copies of the YYRR box in each of three Drosophila species (melanogaster, pseudoobscura, and virilis) and conservation of genomic location within species. Similar analysis of human and rat DNA indicates the presence of YYRR related sequences in mammals as well. YYRR boxes have been localized to two genetic loci in Drosophila: Gld and a gene tentative identified as ted. These two genes exhibit correlated patterns of developmental expression and an identical mutant phenotype. Sequence analysis of the Gld YYRR box in three Drosophila species revealed a high degree of conservation despite its intronic location.     

T7 RNA聚合酶基因表达系统在鱼腥藻7120中构建及hG-CSF的表达

外源基因的表达效率低是蓝藻基因工程发展的瓶颈之一,T7RNA聚合酶表达系统实现了大肠杆菌中外源基因的高效表达,蓝藻与大肠杆菌同为革兰氏阴性菌,具有较高的遗传同源性,在蓝藻中构建T7 RNA聚合酶表达系统有可能提高外源基因在蓝藻中的表达效率。为了在鱼腥藻7120中构建T7RNA聚合酶表达系统,采用重叠延伸PCR技术和酶切连接等方法构建能够表达T7 RNA聚合酶的定点整合载体pEASY-T1-F1-TacT7RNAPCmR-F2以及由T7启动子驱动hG-CSF基因表达的穿梭表达载体pRL-T7-hG-CSF;采用电击转化法将定点整合载体导入野生型鱼腥藻中,通过三亲接合的方法将穿梭表达载体转入已定点整合T7RNA聚合酶的转基因鱼腥藻中。利用PCR技术鉴定外源基因在蓝藻中的存在;RT-PCR方法检测外源基因在蓝藻中的转录情况;Westernblotting实验检测外源基因在蓝藻中的蛋白表达情况。结果表明两种载体构建成功,T7RNA聚合酶基因和hG-CSF基因被转入鱼腥藻中,两个基因均在藻中表达,T7 RNA聚合酶表达系统在鱼腥藻中构建成功,与传统蓝藻表达系统相比,文中在鱼腥藻中构建的T7表达系...     

Signal transduction pathways involved in the expression of the uridine diphosphoglucose pyrophosphorylase gene of Dictyostelium discoideum

The uridine diphosphoglucose pyrophosphorylase (UDPGP1) gene of Dictyostelium discoideum is an excellent marker to study the pathways that control the expression of genes during development. We have previously shown that the UDPGP1 gene is regulated by exogenous cAMP acting on cell-surface cAMP receptors. Various steps in the signal transduction pathway between receptor stimulation and the induction of the gene can now be studied. Induction does not require the synthesis of intracellular cAMP, but does require new protein synthesis. By deletion and transformation with altered genes, two cis-acting sequences that are required for UDPGP1 expression have been identified. A GC-rich palindromic sequence located between -410 and -374 is essential for induction of the gene by extracellular cAMP, but not for its basal expression. A sequence element located between -374 and -337 is required for any basal expression of this gene. When the polarity of the palindromic sequence was reversed such that it resembled the H2K enhancer element, the gene could still be induced by exogenous cAMP. Two DNA binding activities were detected in gel mobility shift assays using a fragment containing both of the regulatory sequence elements of UDPGP1 gene. Transformation with a vector that resulted in the synthesis of anti-sense UDPGP1 RNA led to almost total elimination of the enzyme antigen and no detectable enzyme activity. However, these transformants developed normally, indicating that either UDPGP is not required for development or residual synthesis of UDPGP may be sufficient for normal development.     

Single stranded DNA SP6 promoter plasmids for engineering mutant RNAs and proteins: synthesis of a ''stretched'' preproparathyroid hormone.

The intergenic region of bacteriophage f1 has been subcloned into the bacteriophage SP6 promoter plasmids, pSP64 and pSP65, in both orientations. Coinfection of E. coli with these SP6 promoter/phage f1 chimeric plasmids and the interference resistance phage, IR1, results in the replication and secretion of the pSP6.f1 plasmids as single stranded DNA. Bovine preProPTH cDNAs in both the native form and a form containing an insertion of 117 base pairs in the protein coding region have been inserted in these plasmids. The RNA transcribed from the SP6.f1/preProPTH cDNA constructs was efficiently translated in the wheat germ or reticulocyte cell free systems without addition of a 7-methylguanosine cap to the RNA. In the presence of dog pancreatic or chicken oviduct microsomal membranes, conversion of the resultant pre-proteins to pro-proteins was observed. Confirmation of the "mutated" preProPTH cDNA was determined by dideoxyribonucleotide DNA sequencing of single stranded plasmid DNA. These vectors are suitable for the efficient biosynthesis of large amounts of single or double stranded DNA, and translationally active RNA. The combined properties of single stranded DNA replication and the SP6 promoter simplify the engineering of mutant RNAs and their corresponding proteins. In addition, single stranded DNA or RNA corresponding to either complementary strand may be synthesized as nucleic acid hybridization probes.     

Development and optimization of herpes simplex virus vectors for multiple long-term gene delivery to the peripheral nervous system

Herpes simplex virus (HSV) has often been suggested as a suitable vector for gene delivery to the peripheral nervous system as it naturally infects sensory nerve terminals before retrograde transport to the cell body in the spinal ganglia where latency is established. HSV vectors might therefore be particularly appropriate for the study and treatment of chronic pain following vector administration by relatively noninvasive peripheral routes. However parameters allowing safe and efficient gene delivery to spinal ganglia following peripheral vector inoculation, or the long-term expression of delivered genes, have not been comprehensively studied. We have identified combinations of deletions from the HSV genome which allow highly efficient gene delivery to spinal dorsal root ganglia (DRGs) following either footpad or sciatic nerve injection. These vectors have ICP34.5 deleted and have inactivating mutations in vmw65. We also report that peripheral replication is probably necessary for the efficient establishment of latency in vivo, as fully replication-incompetent HSV vectors allow efficient gene expression in DRGs only after peripheral inoculation at a high virus dose. Very low transduction efficiencies are otherwise achieved. In parallel, promoters have been developed that allow the long-term expression of individual or pairs of genes in DRGs by using elements from the latently active region of the virus to confer a long-term activity onto a number of promoters which otherwise function only in the short term. This work further defines elements and mechanisms within the latently active region that are necessary for long-term gene expression and for the first time allows multiple inserted genes to be expressed from HSV vectors during latency.