Isolation of a cloned DNA segment containing a ribosomal protein gene of Drosophila melanogaster

A Drosophila genomic DNA library in the vector Charon 4 was screened using cDNA derived from the small (6S-12S) poly(A)+ mRNA of 2-6-h-old Drosophila embryos. This fraction of mRNA is enriched for ribosomal protein-coding sequences. The selected recombinants were hybridized to total mRNA under conditions which allowed for isolation of homologous mRNAs. The mRNA from these RNA/DNA hybrids was eluted and translated in vitro. The translation products were analyzed by one- and two-dimensional electrophoresis with authentic ribosomal proteins as standards. One cloned DNA segment was found to contain a ribosomal protein gene, and a sequence which hybridizes strongly to at least 5 other ribosomal protein mRNAs.     

A novel yeast gene coding for a putative protein kinase

A yeast gene termed YKR2 coding for a putative protein kinase was isolated using the cloned cDNA for rabbit protein kinase C as a hybridization probe. The encoded protein YKR2, containing 677 amino acids, shows about 40% sequence identity in the kinase region to a family of serine/threonine-specific protein kinases from various species.     

The mouse Fgfrl1 gene coding for a novel FGF receptor-like protein

The mouse Fgfrl1 gene codes for a novel cell surface protein that is closely related to the family of the FGF receptors. It contains three extracellular Ig C2 loops and an acidic box, which share 29-33% sequence identity (48-50% similarity) with FGF receptors 1-4. The intracellular portion of the novel protein, however, lacks a tyrosine kinase domain required for signal transduction by transphosphorylation. The gene for Fgfrl1 comprises six exons and is located on mouse chromosome 5 in close proximity to the Idua gene for L-iduronidase.     

The nucleotide sequence of the gene coding for Drosophila melanogaster yolk protein 3

The entire sequence of the Drosophila melanogaster yolk protein 3 (YP3) gene (yp3), including 1822 nucleotides (nt) of 5'- and 834 nt of 3'-flanking DNA, has been determined. In addition, the 5' and 3' ends of the mRNA and the two introns have been mapped. The predicted amino acid sequence of YP3 has considerable homology (43%) to the other two yolk proteins of D. melanogaster. The nucleotide sequence of yp3 was compared to the other two yolk protein genes which have the same developmental pattern of expression. In addition to extensive homology between the protein coding regions, we found two small regions of homology between yp3 flanking sequences and a segment of DNA required for normal expression of the yolk protein 1 gene in adult female fat bodies.     

Isolation and characterization of a novel human gene (HFB30) which encodes a protein with a RING finger motif.

A human cDNA, HFB30, encoding a novel protein that contains a RING finger (C3HC4-type zinc finger) motif was isolated. This cDNA clone consists of 3056 nucleotides and encodes an open reading frame of a 474 amino acid protein. From RT-PCR analysis, the messenger RNA was ubiquitously expressed in various human tissues. The gene was located to the chromosome 5q23.3-q31.1 region by PCR-based analyses with both a human/rodent monochromosomal hybrid cell panel and a radiation hybrid mapping panel. Furthermore, the gene consists of nine exons that span about 20 kb of genome DNA.     

Ermap, a gene coding for a novel erythroid specific adhesion/receptor membrane protein

Ermap (erythroid membrane-associated protein), a gene coding for a novel transmembrane protein produced exclusively in erythroid cells, is described. It is mapped to murine Chromosome 4, 57 cM distal to the centromere. The initial cDNA clone was isolated from a day 9 murine embryonic erythroid cell cDNA library. The predicted peptide sequence suggests that ERMAP is a transmembrane protein with two extracellular immunoglobulin folds, as well as a highly conserved B30.2 domain and several phosphorylation consensus sequences in the cytoplasmic region. ERMAP shares a high homology throughout the entire peptide with butyrophilin, a glycoprotein essential for milk lipid droplet formation and release. A GFP-ERMAP fusion protein was localized to the plasma membrane and cytoplasmic vesicles in transiently transfected 293T cells. Northern blot analysis and in-situ hybridization demonstrated that Ermap expression was restricted to fetal and adult erythroid tissues. ERMAP is likely a novel adhesion/receptor molecule specific for erythroid cells.     

ARHGAP10, a novel human gene coding for a potentially cytoskeletal Rho-GTPase activating protein

Rho-GTPase activating proteins (Rho-GAPs) are negative regulators of Rho-GTPase signaling pathways related to actin cytoskeleton dynamics, cell proliferation, and differentiation. We have identified a novel human gene, termed ARHGAP10, that codes for a 1957-aminoacid Rho-GAP, containing a PDZ, a PH, and a Rho-GAP domain. The cDNA is 7118 bp long and has an open reading frame of 5874 bp. A computational analysis located this gene on chromosome 10 band 10p12.32 suggesting that it is composed of 25 exons. Northern analysis revealed that it is widely expressed, with high levels in brain and muscle. Real-time quantitative PCR analysis confirmed an increase in ARHGAP10 expression during differentiation of HL-60 cells with all-trans-retinoic acid and hematopoietic stem cells with erythropoietin, suggesting that this gene could play a role in normal hematopoiesis. The fact that this gene is highly expressed in muscle and brain, which are highly differentiated tissues, further supports the hypothesis that ARHGAP10 is important for cell differentiation.     

A gene coding for a novel protein specific to the olfactory rosettes of Atlantic salmon

We have isolated a 1.6 kb clone from a cDNA library made from the olfactory rosettes of the Atlantic salmon (Salmo salar). The clone contains a 1200 bp, open reading frame (named OSC) which codes for a protein with 400 amino-acid residues (Oscp). The mRNA corresponding to OSC is strongly expressed in the olfactory rosettes and weakly expressed in gills but is expressed in only these two tissues. This suggests that Oscp may have a specific and important role in olfaction. The sequence of Oscp suggests that it is not globular. Predictions show only a small fraction of alpha-helix. Oscp is hydrophilic but with the number of positively charged residues equal to the number of negatively charged residues. No closely similar protein can be found on the basis of homology searches or hydrophobicity comparisons. However, a 44 residue segment (G300 through K343) is significantly homologous to a segment of alpha-lactalbumin (G51 through K94). The similarities include the 19 residues of the "alpha- lactalbumin-lysozyme C signature," the ten residues of the Ca(2+) binding elbow and the four cysteine residues which provide two key disulfide links in alpha-lactalbumin and lysozyme C. Two more Cys residues are also very similarly placed. We conclude that the gene OSC codes for a unique protein which most likely contains a specific site for binding Ca(2+) and plays a unique role in the signal pathway of olfaction in salmon.     

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.     

Identification of a human cDNA sequence which encodes a novel membrane-associated protein containing a zinc metalloprotease motif.

We report the cloning and characterization of a human cDNA predicted to encode a novel hydrophobic protein containing four transmembrane domains and a zinc metalloprotease motif, HEXXH, between the third and fourth transmembrane domains, and have named the molecule metalloprotease-related protein-1 (MPRP-1). The MPRP-1 gene was localized to chromosome 1-p32.3 by radiation hybrid mapping, and Northern blot analysis revealed expression in many organs, with strong expression in the heart, skeletal muscle, kidney and liver. Immunohistochemical analyisis showed that MPRP-1 was localized in the endoplasmic reticulum (ER), and not in the Golgi compartment. Fragments of DNA encoding a segment homologous to the HEXXH motif of MPRP-1 are widely found in bacteria, yeast, plants, and animals. These results suggest that the MPRP-1 may have highly conserved functions, such as in intracellular proteolytic processing in the ER.     

Isolation of a novel protein kinase-encoding gene from yeast by oligodeoxyribonucleotide probing

We have identified a novel protein kinase-encoding gene, KIN3, in the genome of the budding yeast Saccharomyces cerevisiae. The gene was isolated from a library of cloned genomic fragments by probing with an oligodeoxyribonucleotide mixture corresponding to part of a highly-conserved region in the catalytic domain of protein serine-threonine kinases. KIN3 is unique in the yeast genome, maps to chromosome VI and is actively expressed in mitotically dividing cells to produce a 1400 nucleotide (nt) message. The nt sequence of KIN3 predicts a protein product of 43.4 kDa which contains all of the conserved elements found in known protein serine-threonine kinases, although the organisation of these elements in the KIN3 gene product differs significantly from the consensus. The function of the KIN3-encoded protein kinase is unclear although it appears not to be essential for growth, conjugation or sporulation.     

Isolation and characterization of a Drosophila neuropeptide gene

We have purified a 9 amino acid amidated neuropeptide, DPKQDFMRFamide, from whole adult D. melanogaster. This peptide exhibits sequence homology to the molluscan bioactive tetrapeptide FMRFamide and is a novel member of the FMRFamide peptide family. The gene encoding DPKQDFMRFamide has been cloned and characterized. It is present in a single copy per haploid genome, is expressed as a unique 1.7 kb mRNA species, and cytologically maps to 46C on the right arm of chromosome 2. Characterization of a cDNA clone indicates that the precursor protein is 347 amino acids in length and contains 5 copies of DPKQDFMRFamide, as well as 10 additional amidated peptides exhibiting varying degrees of structural relatedness. The Drosophila DPKQDFMRFamide gene and the Aplysia FMRFamide gene are ancestrally related; however, peptides display a higher degree of homology within a species than between species, suggesting intragenic concerted evolution of these neuropeptides.