Reassessment of 79B actin gene expression in the abdomen of adult Drosophila melanogaster

The expression pattern of the 79B actin gene in Drosophila melanogaster has been inferred previously by means of a reporter gene in which 79B actin promoter sequences drive the lacZ coding sequences. Although the 79B actin gene is expressed primarily in muscles of the thorax and first abdominal segment of the adults of both sexes, expression in the remaining abdominal segments appears limited to the male genital muscles and the male-specific Muscle of Lawrence (MOL). This reported abdominal expression pattern has been reassessed. By varying parameters of tissue preparation and lacZ reporter gene detection, expression of the 79B actin gene has been revealed in most dorsal abdominal longitudinal fibres and genital muscles of both females and males. These new results suggest that there are differences in the level of 79B actin gene expression among the various abdominal muscles of both sexes, and that abdominal expression is not limited primarily to male sex structures.     

A cytoskeletal actin gene in the mosquito Anopheles gambiae

Five actin genes have been identified in the mosquito Anopheles gambiae , and a constitutively expressed actin gene has been chosen for detailed analysis. We have physically mapped and sequenced this gene and six associated cDNAs, including translated coding regions, as well as the 5 and 3 flanking sequences. Analysis of stage-specific RNA shows this gene to be present in all stages of mosquito development and in an established A. gambiae cell line, thus indicating a cytoskeietal actin. In the sequence of the translated coding region and in pattern of expression, this gene is very similar to the cytoskeietal actin genes of Droso-phila melanogaster , and in sequence, equally similar to the Artemia cytoskeietal actin gene 403 (99.2% identity among the three amino acid sequences). Sequencing of this A. gambiae actin gene (designated actWior its location in chromosome division 1D) and selected cDNAs shows that it possesses three alternative leader sequences; thus the gene appears to have three alternative promoters. These promoters should ultimately prove useful in the production of transgenic constructs for constitutive expression.     

Characterization of the Drosophila melanogaster retinin gene encoding a cornea-specific protein

Two-dimensional analysis of head extracts from Drosophila melanogaster identified the four eye-specific protein spots corresponding to the retinin protein. The retinin protein spots were specifically stained with phosphoprotein-specific dye, suggesting that the retinin protein undergoes post-translational modification by phosphorylation. Northern blot analysis showed that the retinin gene begins to be expressed during the late stage of puparium formation during development. Analysis of the N-terminal sequence and expression of the retinin gene in S2 suggest that retinin is a secretory protein. Transgenic flies with knockdown expression of the retinin gene by RNA interference (RNAi) were established. However, no significant phenotypic changes in eye structure or phototransduction were observed in the transgenic flies. Western blot analysis and immunohistochemical studies of D. melanogaster eyes suggest that retinin is a cornea-specific protein.     

Reconstitution of human Fc gamma RIII cell type specificity in transgenic mice

The human low affinity receptors for the Fc domain of immunoglobulin G, Fc gamma RIII, are encoded by two genes (IIIA and IIIB) which share >95% sequence identity in both coding and flanking sequences. Despite this extraordinary sequence conservation, IIIA is expressed in natural killer (NK) cells and macrophages and is absent in neutrophils, whereas IIIB is expressed only in neutrophils. To determine the molecular basis for this differential expression, we have generated transgenic mice using the genomic sequences of IIIA and IIIB. IIIA and IIIB transgenic mice show faithful reconstitution of this human pattern of cell type specificity. To determine the cis acting sequence elements that confer this specificity, we constructed chimeric genes in which 5.8 kb of 5' sequences of the IIIB gene has been replaced with a homologous region from the IIIA gene, and conversely, IIIA 5' sequences have been substituted for the analogous region of the IIIB gene. Promoter swap transgenic mice that carry IIIA 5' flanking sequences express Fc gamma RIII in macrophages and NK cells. In contrast, promoter swap transgenic mice that contain IIIB 5' sequences express Fc gamma RIII in neutrophils only. These studies define the elements conferring the cell type-specific expression of the human Fc gamma RIII genes within the 5' flanking sequences and first intron of the human Fc gamma RIIIA and Fc gamma RIIIB genes.     

cDNA cloning,characterization and gene expression of nitric oxide synthase from the silkworm,Bombyx mori

Molecular cloning and nucleotide sequencing of cDNA encoding Bombyx mori nitric oxide synthase (BmNOS) was conducted to analyse its possible role in insect immunity. The amino acid sequence deduced from the BmNOS cDNA showed 84%, 54% and 53% identity with those of NOSs from Manduca sexta, Drosophila melanogaster and Rhodonius prolixus. Recombinant BmNOS produced in insect cells using baculovirus was found to require NADPH, Ca2+, calmodulin and tetrahydrobiopterin (BH4) for its activity. The BmNOS gene was constitutively expressed at a low level in the larval fat body, haemocyte, Malpighian tubule and midgut, and adult antenna, and induced strongly in the fat body by lipopolysaccharide (LPS), suggesting that the BmNOS gene plays different physiological roles in different tissues. Injection of NO donors that produce NO in vivo induced the gene expression of an antibacterial peptide, cecropin B, strongly suggesting that NO produced by BmNOS following LPS stimulation is involved in signal transduction as a signalling molecule for immune gene expression.     

A muscle-specific actin gene from the Mediterranean fruit fly, Ceratitis capitata

A characterization of an actin gene isolated from the genome of the Mediterranean fruit fly, Ceratitis capitata , including the complete sequencing of the coding, 3' and 5' flanking regions of this gene and a partial cDNA was carried out. The partial cDNA was derived from the 3' untranslated region of the actin gene described here, and has been used to identify this gene uniquely. The DNA sequence data presented here, together with the pattern of expression exhibited by this gene during development, strongly support the interpretation that this is a muscle-specific actin gene. Peaks of expression are seen in tissues and during temporal phases of development where muscle differentiation is occurring. The derived protein sequence of the Medfly actin gene shows the highest degrees of similarity, 98.4 and 96.6% respectively, with the two muscle-specific actin genes 79B and 88F from D. melanogaster . The Medfly actin gene also has a single intervening sequence, and an intron is found at the same position in the 79B and 88F actin genes. In the coding region at the DNA level, 17.2 and 16.4% nucleotide differences, respectively, are observed between the Medfly actin gene and these same two D. melanogaster actin genes. The disparity between the amino acid and nucleotide comparisons can be explained, in part, by a high level of synonymous changes in the DNA sequence. In addition, despite the many similarities, codon usage appears to be very different between the actin genes of these species.     

Cloning and expression analysis of takeout/JHBP family genes of silkworm, Bombyx mori

A Bombyx EST cDNA database was searched using the Drosophila takeout gene and nine cDNAs were obtained. The homology search suggested that these genes are widespread in insects and organize a large gene family, and that they have hydrophobic ligands. A phylogenetic tree indicated that the genes are first divided into two large groups, juvenile hormone binding protein and other protein genes, and the latter group diversified within a short time at an early stage. The expression study of five Bombyx genes indicated that they are expressed in various tissues and are regulated by development and feeding conditions. The Bombyx genes might have roles related to the regulation of metabolism, growth or development related to nutritional conditions.     

Molecular cloning and expression of the dihydrofolate reductase (DHFR) gene from adult buffalo fly (Haematobia irritans exigua): effects of antifolates

The folate analogues methotrexate, aminopterin and pyrimethamine were toxic when fed in a blood meal to adult buffalo flies (Haematobia irritans exigua), but aminopterin caused greater mortality than methotrexate, while trimethoprim was not toxic to adult flies. This is the first recorded instance of mortality in adult insects caused by ingestion of folate analogues. In order to investigate the mechanism of this toxicity, the dihydrofolate reductase (DHFR) gene was cloned from adult buffalo fly cDNA using a PCR-based approach. The full-length DHFR coding sequence (BF-DHFR) was 887 bp and contained an open reading frame encoding a protein of 188 amino acids. The deduced protein sequence identities between BF-DHFR and the other known insect DHFR sequences were: Drosophila melanogaster, 75%; Aedes albopictus, 54%; Heliothis virescens, 43%. The BF-DHFR gene has a single 52 bp intron, an organization more similar to Dipteran species (Drosophila and Aedes). The cDNA encoding BF-DHFR was inserted into an Escherichia coli expression vector and the recombinant protein was expressed to levels representing about 25% of total cell protein. The active enzyme was purified by affinity chromatography on methotrexate-agarose and displayed a relatively low affinity (IC50 = 30 nm) for methotrexate.     

Honey bee promoter sequences for targeted gene expression

The honey bee, Apis mellifera, displays a rich behavioural repertoire, social organization and caste differentiation, and has an interesting mode of sex determination, but we still know little about its underlying genetic programs. We lack stable transgenic tools in honey bees that would allow genetic control of gene activity in stable transgenic lines. As an initial step towards a transgenic method, we identified promoter sequences in the honey bee that can drive constitutive, tissue‐specific and cold shock‐induced gene expression. We identified the promoter sequences of Am‐actin5c, elp2l, Am‐hsp83 and Am‐hsp70 and showed that, except for the elp2l sequence, the identified sequences were able to drive reporter gene expression in Sf21 cells. We further demonstrated through electroporation experiments that the putative neuron‐specific elp2l promoter sequence can direct gene expression in the honey bee brain. The identification of these promoter sequences is an important initial step in studying the function of genes with transgenic experiments in the honey bee, an organism with a rich set of interesting phenotypes.     

The cis-regulatory sequences required for expression of the Drosophila melanogaster adult cuticle gene ACP65A

Post-embryonic development in insects requires successive molts. Molts are triggered by ecdysteroids, and the nature of the molt (larval, pupal or adult) is determined by juvenile hormones. The genes encoding cuticle proteins are targets of both classes of hormones, and therefore are interesting models to study hormone action at the molecular level. The Drosophila ACP65A cuticle gene is expressed exclusively during the synthesis of the adult exoskeleton, in epidermal domains synthesising flexible cuticle. We have examined the cis -regulatory sequences of ACP65A using phylogenetic comparisons and functional analysis, and find that only about 180 bp are essential, including an 81 bp intron. The restriction of ACP65A expression appears to depend on a strong repression mechanism.     

SV2A基因真核表达质粒构建及其在HEK293T细胞中的表达

目的构建鼠突触囊泡蛋白2A(SV2A)基因的真核表达质粒,并瞬时转染至人胚肾细胞(HEK293T)中,对其表达进行鉴定。方法以APP/PS1双转基因小鼠海马组织的cDNA为模板,扩增得到长2239 bp的SV2A基因编码序列,将此序列插入到真核表达载体p3×Flag-CMV-10多克隆位点区域中,得到真核表达质粒p3×Flag-CMV-10-SV2A,转化后挑取单克隆菌落经双酶切鉴定后送公司测序,将构建成功的重组质粒转染至HEK293T细胞中,利用蛋白质印迹法(Western blot)检测SV2A基因的表达情况。结果成功构建p3×Flag-CMV-10-SV2A重组质粒,并在转染至HEK293T细胞后,验证了相应蛋白表达。结论利用分子克隆技术成功构建了p3×Flag-CMV-10-SV2A真核表达质粒并在HEK293T细胞中正确表达,为后续实验奠定了基础。     

The Anopheles gambiae alpha-tubulin-1b promoter directs neuronal, testes and developing imaginal tissue specific expression and is a sensitive enhancer detector

A knowledge gap in mosquito functional genetic analysis is the dearth of characterized regulatory regions that can target tissue specific transgene expression. To broaden the tools available, a promoter region of the Anopheles gambiaeα-tubulin1b gene has been assayed following fusion to the green fluorescent protein (GFP) reporter gene and stable transformation of An. gambiae. In eight transgenic lines, the Angtub α1b regulatory region directed a core profile of tissue specific expression in the head, chordotonal organs, ventral nerve cord and testes. This profile overlaps those seen for α2-tubulin expression in Drosophila melanogaster and Bombyx mori. In addition, widespread position dependant expression was observed in other specific tissues that were unique to each line. For example, in different lines, expression was observed in larval and adult muscles, fatbody, cuticle and midgut secretory cells. The majority of genomic transgene insertions were mapped to within 10 kb of a gene, suggesting that the Angtub α1b basal promoter is particularly sensitive to enhancers and may be suitable to form the basis of a sensitive enhancer trapping construct, in combination with a binary expression system such as Gal4-UAS.     

Identification and function of Abdominal-A in the silkworm, Bombyx mori

Abdominal-A ( adb-A ) is a key gene in the development of insects. To understand its function in the silkworm, we cloned 1193 bp of the abd-A gene of Bombyx mori ( Bmabd-A ), including the complete coding sequence and part of the 3' untranslated region sequence. Bmabd-A has at least three mRNA splice variants with coding sequences of lengths 1032, 1044 and 1059 bp, encoding 343, 347 and 352 amino acids, respectively. Each splice variant of Bmabd-A has three exons and differs only in second exon size. Bmabd-A was expressed at low levels in unfertilized eggs, but increased gradually in fertilized eggs after laying 22 h. Bmabd-A expression decreased in ant silkworms (newly hatched silkworms). After RNA interference for Bmabd-A , the embryos had two mutant phenotypes, either completely or partially absent abdominal feet from the third to sixth abdominal segments, suggesting that Bmabd-A is responsible for normal development of the third to sixth abdominal segments during embryonic development.