Cloning of a cDNA encoding a novel putative G-protein-coupled receptor expressed in specific rat brain regions.

A cDNA clone encoding a novel putative G-protein-coupled receptor was isolated from a rat brain cDNA library using a PCR-amplified cDNA fragment as a hybridization probe. The 3,615-bp-long nucleotide sequence predicts a single open reading frame of 1,173 bp coding for 391 amino acids, giving a calculated molecular weight of 42.75 kD. The amino acid sequence shares features common to many other receptors, including the seven membrane-spanning hydrophobic regions and putative asparagine-linked glycosylation and phosphorylation sites. Northern blot analysis reveals that a corresponding approximately 3.7-kb mRNA is expressed in specific brain regions such as hypothalamus, cortex, hippocampus, and thalamus but not in other organs analyzed. Although the ligand for this receptor has not yet been identified, it shares some similarities with the vascular type-1 angiotensin II receptor, the vasoactive intestinal peptide (VIP) receptor, and the chemotactic receptors for human C5a anaphylatoxin and the formyl peptide fMet-Leu-Phe.     

Cloning of a novel G-protein-coupled receptor from the sea anemone nervous system

We describe the cloning and analysis of genomic and cDNA copies of a gene from sea anemones that encodes a new member of the G-protein-coupled receptor family. The receptor shows similarity to previously described receptors for biogenic amines such as adrenaline, serotonin, and octopamine, as well as a variety of small molecule agonists and peptides, although we have been unable to determine which ligand is the natural agonist. Antibodies generated against the recombinant receptor protein identify a single protein with a molecular weight of 66 kDa in membrane preparations. Immunofluorescence studies using the same antibody have enabled localization of the receptor in the nervous system. Western blotting and RT-PCR analysis reveal that a homologue of this receptor is expressed in jellyfish and soft coral. We suggest that the receptor plays a role in neurotransmission in the sea anemone and other members of the phylum Cnidaria.     

Cloning and chromosomal localization of a human endothelin ETA receptor.

A cDNA clone encoding a human endothelin receptor was isolated from a placenta cDNA library. The deduced amino acid sequence of the clone is 94% identical to the bovine endothelin ETA receptor and represents the human homologue. The human endothelin ETA receptor gene was localized to chromosome 4 by analysis of its segregation pattern in rodent-human hybrids.     

Cloning, expression, and chromosomal mapping of a human ganglioside sialidase.

Here we report the cDNA sequence of a human ganglioside sialidase. The cDNA was isolated from a human brain cDNA library by screening with a 240 bp probe generated by polymerase chain reaction using primers based on the sequences of rat cytosolic and bovine membrane sialidases which we previously cloned. The 3.0 kb cDNA encodes an open reading frame of 436 amino acids containing a putative transmenbrane domain and an Arg-Ile-Pro and three Asp-box sequences characteristic of sialidases and showing overall 83% and 39% identities to the bovine and rat enzymes, respectively. Northern blot analysis revealed high expression in skeletal muscle and testis, but low level in kidney, placenta, lung, and digestive organs. Transient expression of the cDNA in COS-1 cells resulted in a 130-fold increase in sialidase activity compared to the control level, and the activity was found to be almost specific for gangliosides. Fluorescent in situ hybridization allowed the human sialidase gene localized to chromosome 11 at q 13.5.     

Cloning and localization of rgpr85 encoding rat G-protein-coupled receptor

In an attempt to isolate genes involved in the brain development using ordered differential display PCR, we cloned rgpr85 which encodes rat G-protein-coupled receptor with high degree of identity to the amine-like neurotransmitter receptors. This gene was found to be localized at rat chromosome 4q21. In situ hybridization demonstrated that rgpr85 was predominantly expressed in the developing brain and spinal cord. Hybridization signal was especially abundant within the embryonic cortical plates where postmitotic cortical neurons are localized. In the cerebral cortex, the expression of rgpr85 was gradually decreased postnatally and became undetectable by P18. However, weak but significant expression of rgpr85 was maintained in the adult hippocampal formation, olfactory bulb, and cerebellum. Interestingly, rgpr85 expression was transiently induced in the adult hippocampal formation, piriform cortex, and amygdaloid complex by kainic acid (KA) treatment. Thus, dynamic regulation of rgpr85 expression suggests an importance of rgpr85-mediated signaling in the development of cerebral cortex and in the KA-induced responses in the adult brain.     

Molecular cloning and chromosomal mapping of CCND genes encoding human D-type cyclins.

A human D-type cyclin gene (CCND1/cyclin D1/PRAD1) was previously isolated by virtue of its ability to complement a triple G1 cyclin (Cln) deficiency of Saccharomyces cerevisiae and was also identified as a candidate BCL1 oncogene. We now report the molecular cloning of two additional human D-type cyclin genes, CCND2 (cyclin D2) and CCND3 (cyclin D3). All three human D-type cyclin genes encode small (33-34 kDa) proteins that share an average of 57% identity over the entire coding region and 78% in the cyclin box. The D-type cyclins are most closely related to cyclin A (39% identity) and cyclin E (36%), followed by cyclin B (29%) and cyclin C (21%). Isolation and characterization of genomic clones revealed two pseudogenes corresponding to CCND2 and CCND3, respectively. All three cyclin D genes are interrupted by an intron at the same position. CCND2 has been mapped to chromosome 12p13, and CCND3 has been mapped to chromosome 6p21.     

Cloning chromosomal lac genes of Klebsiella pneumoniae

The chromosomal gene for beta-galactosidase from Klebsiella pneumoniae strain T17R1 and associated regulatory genes have been cloned as a 5-kb HindIII fragment in the pBR322 plasmid vector. The beta-galactoside permease gene is not present in a functional form in the 5-kb fragment. The K. pneumoniae genes are expressed in an Escherichia coli host. The synthesis of beta-galactosidase is inducible by isopropyl-beta-D-galactosidase (IPTG) and is sensitive to catabolite repression. There appears to be greater homology between the K. pneumoniae and E. coli structural genes for beta-galactosidase than there is between the respective repressor genes.     

Cloning and chromosomal mapping of nuclear genes encoding chloroplast and cytosolic glyceraldehyde-3-phosphate-dehydrogenase from Arabidopsis thaliana

Both cDNA and genomic clones for the nuclear genes encoding chloroplast (cp) (gapA and gapB) and cytosolic (gapC) glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from Arabidopsis thaliana have been isolated and characterized. Genomic Southern-blot analyses indicate that there is only one copy of each gapA, gapB and gapC gene in A. thaliana. Comparison of the deduced amino acid (aa) sequences shows that the A and B subunits are highly similar (80% positional aa identity), while there is less similarity between the cp and cytosolic subunits (45% aa identity). These relationships are consistent with the idea that the cp and cytosolic GAPDHs evolved from different lineages, as suggested in our previous study of tobacco GAPDHs [Shih et al., Cell 47 (1986) 73-80]. In addition, the chromosomal locations for the three gap genes were determined by restriction fragment length polymorphism mapping; the three gap genes are not closely linked, gapA (55.8 cM) and gapC (0.0 cM) are on chromosome 3, and gapB (51.3 cM) is on chromosome 1.