Embryonic expression of three mouse genes with homology to the Drosophila melanogaster prickle gene

The Drosophila melanogaster gene prickle-spiny-legs (pk) functions in an intercellular feedback loop that is central to the establishment of planar cell polarity in the eye and epidermis of the fly, by modulating Frizzled-Disheveled signalling. Here we identify three mouse prickle-related genes (dyxin, testin and prickle) and describe their expression pattern during murine embryogenesis (E7.5-E15.5). We report that the three genes are expressed in restricted areas of the developing mouse brain: dyxin in the most ventral region of the neural tube and in some localized regions of the ventricular layer of the mesencephalon and rhombencephalon, prickle in the pons region, ventrolateral part of rhombencephalon and motoneurons in the spinal cord, and testin in differentiating neurons of the spinal cord and retina. At the stages analyzed, the main site of expression of testin is the migrating cranial neural crest, while the expression of dyxin is noticeable in myotomal cells and its derivatives, with prickle expression being reciprocally localized to some sclerotomal derivatives, like bone primordia. prickle is also expressed in the apical ectodermal ridge and the most distal mesenchyme of the forming limb buds.     

A human gene family with sequence homology to Drosophila melanogaster Hsp70 heat shock genes

A growing literature describes the structure and regulation of prokaryotic and eukaryotic heat shock genes. We here report the isolation of several members of a human heat shock protein 70 (hsp 70) multigene family which contains at least 10 different genes and/or pseudogenes exhibiting sequence homology to the hsp70 gene of Drosophila melanogaster. Eight nonoverlapping recombinant lambda phages from a lambda-Charon4A human genomic library were studied by restriction mapping. One lambda clone was sequenced and characterized as a hsp70 pseudogene inserted into a rearranged human HindIII 1.9-kilobase repeated DNA sequence. This pseudogene is probably located on the X chromosome. Its predicted amino acid sequence shows extensive homology to those of Drosophila hsp70, trout hsp70, Xenopus hsp70, yeast hsp70, and some homology to the heat-inducible dnaK gene product of Escherichia coli. Amino acid homology is clustered, suggesting evolutionary conservation of domains critical to the function of this protein in both prokaryotic and eukaryotic cells.     

Embryonic expression patterns of Drosophila ACS family genes related to the human sialin gene

The anion/cation symporter (ACS) family is a large subfamily of the major facilitator superfamily (MFS) of transporters. ACS family permeases are widely distributed in nature and transport either organic or inorganic anions in response to chemiosmotic cation gradients. The only protein in the ACS family to which a human disease has been linked, is sialin, the proton-driven lysosomal carrier for sialic acid. Genetic defects in sialin cause a lysosomal storage disease in humans. Here we have identified a group of conserved Drosophila ACS family genes related to sialin and studied their expression patterns throughout embryogenesis. Drosophila sialin-related genes are expressed in a wide variety of tissues. Expression is frequently observed throughout various parts of the intestinal tract, including Malpighian tubules and salivary glands. Additionally, some genes are expressed in vitellophages (yolk nuclei), nervous system, respiratory tract and a number of other embryonic tissues. These data will aid the establishment of a fruitfly model of human lysosomal storage disorders, the most common cause of neurodegeneration in childhood.     

Expression during embryogenesis of a mouse gene with sequence homology to the Drosophila engrailed gene

Regions of the mouse and human genomes with strong homology to the Drosophila engrailed gene have been identified by Southern blot analysis. One mouse engrailed-like region, Mo-en.1, has been cloned and partially sequenced; homology with the engrailed gene is localized to a 180 bp engrailed-like homeo box and 63 nucleotides immediately 3' to it. The protein sequence this region can encode includes 81 amino acids, of which 60 (75%) are identical with those of the putative translation product of the corresponding engrailed sequence. These data suggest that Mo-en.1 represents a mouse homolog of a gene of the Drosophila engrailed gene complex. Mo-en.1 has been mapped to chromosome 1, indicating it is not linked to other homeo box sequences that have been mapped in the mouse genome. Analysis of poly(A)+ RNA extracted from teratocarcinoma cells and whole mouse embryos demonstrates that the conserved homeo box region of Mo-en.1 is expressed differentially during mouse embryogenesis.     

Embryonic expression of the period clock gene in the central nervous system of Drosophila melanogaster.

We have examined the temporal and spatial expression of the 4.5-kb mRNA that is transcribed from the period locus of Drosophila melanogaster and is the best candidate for the per gene product. Both Northern blot analyses and hybridizations in situ to tissue sections reveal significant expression of the 4.5-kb mRNA in embryos. This expression is limited to the central nervous system of the developing embryo and is localized within the brain and ventral ganglia. The 4.5-kb mRNA is enriched in adult heads (by Northern blotting) although we were not able to detect specific localization (in situ). In addition to the physiological role the 4.5-kb mRNA might have in maintaining biological rhythms, we now suggest that it has a developmental role for establishing mechanisms that are necessary for eventual expression of clock functions.     

New genes interacting with the zeste gene in Drosophila melanogaster]

We have found that mutations in the enhancer of yellow, 1,2 and 3 loci strongly enhance the effect of zv77h-mutation (full inactivation of the zeste locus) on the white locus expression. Their effect is realized through the distal white enhancer which is located 1,1 kb upstream to the promoter. It is suggested that the protein products of enhancers of yellow 1,2 and 3 represent a family of proteins which, like zeste protein, are responsible for formation of contacts between elements located at a large distance in the genome.     

Structural analysis of the three vitellogenin genes in Drosophila melanogaster.

Genomic fragments coding for sequences expressed as abundant mRNA in female Drosophila melanogaster were isolated from a lambda library. Hybridization of these clones to polytene chromosomes. in situ, identified four which mapped to X chromosomal region 9A to 9B, the locus for yolk proteins 1 and 2 (Ypl,2) and two which mapped to 12A6-7 to 12D3, the locus for Yp3. These clones were mapped with restriction enzymes, and the coding regions and regions of homology determined by Southern blots probed with cDNA, 5'-end-labelled RNA and nick-translated DNA. Heteroduplex and R-loop mapping confirmed that three of the clones carried two genes separated by about 1.4 kb and oriented in opposite directions. Southern blots probed with cDNA made from alkali-hydrolyzed RNA showed that these genes had their 5' ends next to each other. All 3 genes show homology to each other and have a main coding region of about 1.3 kb, the approximate size for the mRNAs.     

Isolation of a mouse DNA fragment with homology to a Drosophila ribosomal protein gene

A mouse genomic library in lambda Charon 4A was screened for putative ribosomal protein genes using a fragment of the gene encoding Drosophila ribosomal protein 49 as a hybridization probe under nonstringent hybridization conditions. A recombinant phage was selected and its restriction enzyme map determined. The major species of mouse poly(A)+ mRNA homologous to the putative gene is about 740 nucleotides long.     

Dual-tagging gene trap of novel genes in Drosophila melanogaster

A gene-trap system is established for Drosophila. Unlike the conventional enhancer-trap system, the gene-trap system allows the recovery only of fly lines whose genes are inactivated by a P-element insertion, i.e., mutants. In the gene-trap system, the reporter gene expression reflects precisely the spatial and temporal expression pattern of the trapped gene. Flies in which gene trap occurred are identified by a two-step screening process using two independent markers, mini-w and Gal4, each indicating the integration of the vector downstream of the promoter of a gene (dual tagging). mini-w has its own promoter but lacks a polyadenylation signal. Therefore, mini-w mRNA is transcribed from its own promoter regardless of the vector integration site in the genome. However, the eyes of flies are not orange or red unless the vector is incorporated into a gene enabling mini-w to be spliced to a downstream exon of the host gene and polyadenylated at the 3' end. The promoter-less Gal4 reporter is expressed as a fusion mRNA only when it is integrated downstream of the promoter of a host gene. The exons of trapped genes can be readily cloned by vectorette RT-PCR, followed by RACE and PCR using cDNA libraries. Thus, the dual-tagging gene-trap system provides a means for (i) efficient mutagenesis, (ii) unequivocal identification of genes responsible for mutant phenotypes, (iii) precise detection of expression patterns of trapped genes, and (iv) rapid cloning of trapped genes.     

Characterization and expression of collagen-like genes in Drosophila melanogaster

We have used a cloned chicken collagen cDNA sequence to help identify hypothetic members of the collagen gene family from Drosophila melanogaster. Several experimental evidences have been obtained which indicate that the Drosophila genome contains numerous collagen-like sequences. We have characterized in more detail ten distinct DNA sequences that hybridized strongly to the heterologous collagen probe. By in situ hybridization we have shown that these sequences are dispersed throughout the Drosophila genome. Two of them are shown to originate from the previously described DCg 1 and DCg 2 collagen genes. In other respects, we show that in addition to DCg 1 and DCg 2, at least five putative collagen genes are expressed during the Drosophila lifetime. These genes are unique, and some of them are seen to be transcribed into different size classes of mRNAs. Additionally, the data presented so far demonstrate that the expression of these genes is regulated temporally and/or quantitatively during the Drosophila life cycle.     

Cloning and characterization of two mouse genes with homology to the yeast Sir2 gene

The yeast Sir2 gene encodes a protein (Sir2p) that plays an essential role in silencing regulation at mating-type loci, rDNA, and telomeres. Recent studies have also shown that the protein participates in cell cycle regulation, DNA double-strand break repair, meiotic checkpoint control, and histone deacetylation. Overexpression of wildtype Sir2p in yeast resulted in an extended life span but mutant Sir2p shortened the life span, suggesting its function in aging processes. Sir2p is evolutionarily conserved from prokaryotes to higher eukaryotes. However, its function(s) in mammals remains unknown. To investigate Sir2p function(s) in mice, we cloned and characterized two mouse Sir2-like genes. Our results revealed that the two mouse Sir2-like proteins (mSIR2L2 and mSIR2L3) are most similar to the human Sir2-like proteins SIR2L2 and SIR2L3, respectively. Sir2 core domains are highly conserved in the two proteins and yeast Sir2p; however, the intracellular localizations of both mSIR2L2 and mSIR2L3 differ from that of yeast Sir2p and from one another. The two mouse genes have completely different genomic structures but were mapped on the same chromosome. It seems that the two mouse proteins, though they have Sir2 conserved domains, may function differently than yeast Sir2p.     

Drosophila melanogaster S2 cells for expression of heterologous genes: From gene cloning to bioprocess development

In the present review we discuss strategies that have been used for heterologous gene expression in Drosophila melanogaster Schneider 2 (S2) cells using plasmid vectors. Since the growth of S2 cells is not dependent on anchorage to solid substrates, these cells can be easily cultured in suspension in large volumes. The factors that most affect the growth and gene expression of S2 cells, namely cell line, cell passage, inoculum concentration, culture medium, temperature, dissolved oxygen concentration, pH, hydrodynamic forces and toxic metabolites, are discussed by comparison with other insect and mammalian cells. Gene expression, cell metabolism, culture medium formulation and parameters involved in cellular respiration are particularly emphasized. The experience of the authors with the successful expression of a biologically functional protein, the rabies virus glycoprotein (RVGP), by recombinant S2 cells is presented in the topics covered.     

He-T family DNA sequences in the Y chromosome of Drosophila melanogaster share homology with the X-linked Stellate genes

The genome of Drosophila melanogaster contains a class of repetitive DNA sequences called the He-T family, which is unusual in being confined to telomeric and heterochromatic regions. The specific He-T fragment designated Dm665 was cloned in yeast by selection for an autonomously replicating sequence (ARS). Dm665 contains a restriction fragment length polymorphism (RFLP) that is specific to males and thus derives from the Y chromosome. Deletion mapping using X-Y translocations indicates that sequences homologous to Dm665 occur in at least one major cluster in each arm of the Y chromosome. Among 20 yeast artificial chromosome (YAC) clones containing Drosophila sequences homologous with Dm665, four clones derive from defined regions of the long arm of the Y and two from the short arm. The sequence of Dm665 is 2443 bp long, consists of 59% A+T, and contains no significant open reading frames or direct or inverted repeats. However, Dm665 contains a region of 650 bp that shares homology with portions of the X-linked locus Stellate.by W. Hennig