The embryonic expression patterns of zfh-1 and zfh-2, two Drosophila genes encoding novel zinc-finger homeodomain proteins.

The zfh-1 and zfh-2 genes of D. melanogaster encode novel proteins containing both homeodomain and C2-H2 zinc-finger DNA-binding motifs. Antisera against these proteins were used to investigate their expression patterns during embryonic development. The zfh-1 gene is expressed in the mesoderm of early embryos and in a number of mesodermally-derived structures of late embryos, including the dorsal vessel, support cells of the gonads, and segment-specific arrays of adult muscle precursors. In addition, zfh-1 is expressed in the majority of identified motor neurons of the developing CNS. The mesodermal zfh-1 expression requires the products of the twist and snail genes. The zfh-2 gene displays a more limited expression pattern, largely restricted to the CNS of late embryos. Ubiquitous zfh-1 expression in transgenic flies bearing an hsp70-zfh-1 construct has specific developmental consequences, including embryonic CNS defects as well as adult eye and bristle abnormalities. The expression patterns of zfh-1 and zfh-2 suggest that both genes may be involved in Drosophila neurogenesis and that zfh-1 may have additional functions in mesoderm development.     

Characterization of two Drosophila melanogaster cytochrome c genes and their transcripts.

Analysis of total Drosophila melanogaster DNA by genomic blot hybridization indicates that two cytochrome c-like sequences exist in the Drosophila genome. These two sequences, DC3 and DC4, have been isolated from a Charon 4A-D. melanogaster genomic library. DC3 and DC4 are located within a 4 kb region of DNA, at position 36A 10-11, on the left arm of chromosome 2. The nucleotide sequence of these two clones has been determined. Both DC3 and DC4 can encode functional cytochrome c proteins. The polypeptide sequences predicted by these two genes, however, differ at 32 amino acid residues. DC4 is expressed at varying, but relatively high levels throughout Drosophila development. In contrast, DC3 is expressed at constant, but relatively low levels throughout development.     

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.     

Registration of the expression patterns of Drosophila segmentation genes by two independent methods

MOTIVATION: To construct an integrated map of Drosophila segmentation gene expression from partial data taken from individual embryos. RESULTS: Spline and wavelet based registration techniques were developed to register Drosophila segmentation gene expression data. As ground control points for registration we used the locations of extrema on gene expression patterns, represented in 1D. The registration method was characterized by unprecedented high accuracy. A method for constructing the integrated pattern of gene expression at cellular resolution was designed. These patterns were constructed for 9 segmentation genes belonging to gap and pair-rule classes.     

Characterization and expression analysis of two human septin genes, PNUTL1 and PNUTL2

The presence and role of septin proteins in yeast is well documented, but there is a growing appreciation for this family of proteins beyond yeast and extending to human cells. In this report we present the characterization and comparison of two highly similar human septin genes, PNUTL1 and PNUTL2. We compare the exon/intron structure of both genes, the steady-state mRNA levels in tumor cell lines and adult organs, the conceptual translation products from alternatively processed mRNAs and the development of specific immunologic reagents distinguishing either PNUTL1 or PNUTL2. The results illustrate a remarkable similarity between the two genes and their protein products while identifying specific differences in mRNA expression patterns. A summary of the described functional roles for mammalian septins is discussed along with an attempt to assimilate the alternative nomenclature existing for the same human septins, such as references to PNUTL1 and PNUTL2 as hCDCrel-1 and hCDCrel-2, respectively. The characterization of PNUTL1 and PNUTL2 represents a fundamental step in completing the characterization of the entire family of human septin genes.     

nos-1 and nos-2, two genes related to Drosophila nanos, regulate primordial germ cell development and survival in Caenorhabditis elegans.

In Drosophila, the posterior determinant nanos is required for embryonic patterning and for primordial germ cell (PGC) development. We have identified three genes in Caenorhabditis elegans that contain a putative zinc-binding domain similar to the one found in nanos, and show that two of these genes function during PGC development. Like Drosophila nanos, C. elegans nos-1 and nos-2 are not generally required for PGC fate specification, but instead regulate specific aspects of PGC development. nos-2 is expressed in PGCs around the time of gastrulation from a maternal RNA associated with P granules, and is required for the efficient incorporation of PGCs into the somatic gonad. nos-1 is expressed in PGCs after gastrulation, and is required redundantly with nos-2 to prevent PGCs from dividing in starved animals and to maintain germ cell viability during larval development. In the absence of nos-1 and nos-2, germ cells cease proliferation at the end of the second larval stage, and die in a manner that is partially dependent on the apoptosis gene ced-4. Our results also indicate that putative RNA-binding proteins related to Drosophila Pumilio are required for the same PGC processes as nos-1 and nos-2. These studies demonstrate that evolutionarily distant organisms utilize conserved factors to regulate early germ cell development and survival, and that these factors include members of the nanos and pumilio gene families.     

Expression domains of the Cf1a POU domain protein during Drosophila development

We have used antibodies directed against a unique portion of the Drosophila POU domain protein Cfla to localize its sites of expression in developing embryos. Cfla protein is first detected during germ band extension in the tracheal placodes and in the midline mesectoderm cells. Tracheal expression continues throughout embryonic development, especially in the main longitudinal tracheal trunks. Additional sites of high Cfla expression are in the anterior portion of the hindgut, the roof of the stomodeum, a subset of central nervous system cells, the oenocytes, and the ring gland. In addition, Cfla expression was localized in embryos mutant for several loci involved in determining fate along the midline of the CNS and the tracheal system. Cfla midline cell expression is dependent on proper single-minded gene function, and Cfla either regulates or acts in parallel to the genes pointed and rhomboid during midline CNS and tracheal development.     

Characterization of two related Drosophila gamma-tubulin complexes that differ in their ability to nucleate microtubules

gamma-tubulin exists in two related complexes in Drosophila embryo extracts (Moritz, M., Y. Zheng, B.M. Alberts, and K. Oegema. 1998. J. Cell Biol. 142:1- 12). Here, we report the purification and characterization of both complexes that we name gamma-tubulin small complex (gammaTuSC; approximately 280,000 D) and Drosophila gammaTuRC ( approximately 2,200,000 D). In addition to gamma-tubulin, the gammaTuSC contains Dgrip84 and Dgrip91, two proteins homologous to the Spc97/98p protein family. The gammaTuSC is a structural subunit of the gammaTuRC, a larger complex containing about six additional polypeptides. Like the gammaTuRC isolated from Xenopus egg extracts (Zheng, Y., M.L. Wong, B. Alberts, and T. Mitchison. 1995. Nature. 378:578-583), the Drosophila gammaTuRC can nucleate microtubules in vitro and has an open ring structure with a diameter of 25 nm. Cryo-electron microscopy reveals a modular structure with approximately 13 radially arranged structural repeats. The gammaTuSC also nucleates microtubules, but much less efficiently than the gammaTuRC, suggesting that assembly into a larger complex enhances nucleating activity. Analysis of the nucleotide content of the gammaTuSC reveals that gamma-tubulin binds preferentially to GDP over GTP, rendering gamma-tubulin an unusual member of the tubulin superfamily.     

Unique patterns of androgen regulation of the expression of two genes in murine kidney

The kidneys of androgen stimulated mice exhibit a hypertrophic response but no hyperplasia or concomitant DNA replication. Androgens increase the expression of several genes in mouse kidney. The response of the beta-glucuronidase gene to testosterone in this tissue is characterized by a 1-2 day lag and relatively slow induction kinetics. The gene coding for kidney androgen-regulated protein (KAP) exhibits quite a different response to the hormone when compared on the basis of initial response to a given dose, dose required to produce maximal response, and apparent sensitivity to low levels of androgen-receptor complexes in renal nuclei. The analysis of the accumulation of the mRNAs produced by these two genes suggests that gene-specific differential sensitivity to androgen receptor complexes governs the development of the cellular male phenotype in this tissue.     

Drosophila melanogaster genes for U1 snRNA variants and their expression during development.

We have cloned and characterized a complete set of seven U1-related sequences from Drosophila melanogaster. These sequences are located at the three cytogenetic loci 21D, 82E, and 95C. Three of these sequences have been previously studied: one U1 gene at 21D which encodes the prototype U1 sequence (U1a), one U1 gene at 82E which encodes a U1 variant with a single nucleotide substitution (U1b), and a pseudogene at 82E. The four previously uncharacterized genes are another U1b gene at 82E, two additional U1a genes at 95C, and a U1 gene at 95C which encodes a new variant (U1c) with a distinct single nucleotide change relative to U1a. Three blocks of 5' flanking sequence similarity are common to all six full length genes. Using specific primer extension assays, we have observed that the U1b RNA is expressed in Drosophila Kc cells and is associated with snRNP proteins, suggesting that the U1b-containing snRNP particles are able to participate in the process of pre-mRNA splicing. We have also examined the expression throughout Drosophila development of the two U1 variants relative to the prototype sequence. The U1c variant is undetectable by our methods, while the U1b variant exhibits a primarily embryonic pattern reminiscent of the expression of certain U1 variants in sea urchin, Xenopus, and mouse.     

Characterization of two genes, Impa1 and Impa2 encoding mouse myo-inositol monophosphatases.

The enzyme myo-inositol monophosphatase (Impa) catalyzes the synthesis of free myo-inositol from various myo-inositol monophosphates in the phosphatidylinositol signaling system. Impa is a lithium-blockable enzyme that has been hypothesized to be the biological target for lithium-salts used as mood-stabilizing drugs in the treatment of manic-depressive (bipolar) illness. As an initial step to explore the functional consequences of reduced or absent Impa activity in an animal model we here report the isolation of two Impa-encoding mouse genes, Impa1 and Impa2. Impa1 spans approximately 17.5 kb and contains nine exons of 46--1354 bp encoding a protein of 277 amino acids. Impa2 spans at least 19.5 kb and contains eight exons of 46--444 bp size encoding a protein of 290 amino acids. The genomic structure including the positions of the exon-intron splice sites seems to be conserved among myo-inositol monophosphatase genes in mammalian species. One or more Impa-like genes do also exist in evolutionary more distant species like invertebrates, plants and bacteria. The proteins encoded by the non-vertebrate genes seem to be equally related to Impa1 and Impa2. We therefore suggest that the Impa1 and Impa2 genes duplicated from a common ancestral gene after the evolutionary divergence of vertebrates.     

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.     

Polymorphism patterns in two tightly linked developmental genes,Idgf1 and Idgf3, of Drosophila melanogaster

A new developmental gene family, recently identified in D. melanogaster, has been called imaginal disc growth factors (IDGF) because the proteins promote growth of cell lineages derived from imaginal discs. These are the first genes reported that encode polypeptide factors with mitotic activity in invertebrates. Characteristics such as similar arrangement of introns and exons, small size, and different cytological localization make this family an excellent candidate for evolutionary studies. We focus on the loci Idgf1 and Idgf3, two genes that possess the most distinctive features. We examine the pattern of intra- and interspecific nucleotide variation in the sequences from 20 isogenic lines of D. melanogaster and sequences from D. simulans and D. yakuba. While MK, HKA, and Tajima's tests of neutrality fail to reject a neutral model of molecular evolution, Fu and Li's test with outgroup and McDonald's test suggest that balancing selection is modulating the evolution of the Idgf1 locus. The rate of recombination between the two loci is high enough to uncouple any linkage disequilibrium arising between Idgf1 and Idgf3, despite their close physical proximity.     

Characterization and developmental expression of beta tubulin genes in Drosophila melanogaster.

Genomic clones containing beta tubulin sequences were isolated from a lambda library of Drosophila melanogaster. In situ hybridization localized three genes to 56D and 60B on chromosome 2 as well as to 85D on chromosome 3. The latter was known through genetic analysis to be specifically expressed during spermatogenesis. The genomic clone, pTu85, derived from this region contains one complete beta tubulin coding region as well as the 3' end of an additional so far unidentified beta tubulin gene. Genomic Southern hybridizations reveal a total of five fragments with beta tubulin homology. Clone pTu56 codes for an RNA of 1.8 kb which is expressed in all developmental stages. Clone pTu60 codes for a 2.5-kb RNA expressed during embryogenesis and pupation. In testes RNA we detected a 2.2-kb message homologous to pTu85.