First-generation monoclonal antibodies identifying the human leukotriene B(4) receptor-1

The leukotriene B(4) receptor (BLTR) is a seven-transmembrane chemoattractant receptor that is important in pro-inflammatory responses. We have produced the first widely applicable monoclonal antibodies against the human BLTR and confirmed the antibody specificity using flow cytometric analysis of three different cell lines stably expressing the recombinant receptor. The antibodies did not cross-react with the recently cloned second LTB(4) receptor, BLTR2, or the Cys LT1 and Cys LT2 receptors. Functional analysis in combination with two-color flow cytometry showed that the BLTR antibodies bind to cells that are activated by LTB(4). The antibodies were shown to recognize BLTR in cell ELISA and immunocytochemistry. Endogenous expression of BLTR in CD15-positive blood leukocytes and in differentiated HL-60 cells was also demonstrated with the antibodies.     

A Cluster of Four Novel Human G Protein-Coupled Receptor Genes Occurring in Close Proximity to CD22 Gene on Chromosome 19q13.1

In our search for novel human galanin receptor (GALR) subtypes, human genomic DNA was PCR amplified using sets of degenerate primers based on conserved sequences in human and rat GALR. The sequence of one of the subcloned PCR products revealed homology to a sequence in the 3′ region of the human CD22 gene following a BLAST search of GenBank's database. A search for open reading frames (ORF) in the non-coding CD22 sequence resulted in identification of two novel putative intronless genes, GPR40 and GPR41. The recent submission of sequence overlapping the downstream CD22 sequence revealed a possible polymorphic insert containing a third intronless gene, GPR42, sharing 98% amino acid identity with GPR41, followed by a fourth intronless gene, GPR43. Thus, the GPR40, GPR41, GPR42, and GPR43 genes, respectively, occur downstream from CD22, a gene previously localized on chromosome 19q13.1. The four putative novel human genes encode new members of the GPCR family and share little homology with GALR.     

Mammalian keratin gene families: organisation of genes coding for the B2 high-sulphur proteins of sheep wool.

We have isolated two genomic clones containing three B2 high-sulphur keratin genes from a sheep genomic library constructed in Charon 4A. These genes do not contain intervening sequences. Two genes, encoding the B2A and B2D proteins are closely linked in the genome, being separated by 1.9 kb, and are transcribed in the same direction. Although there is extensive sequence conservation in the 5' non-coding and coding regions, the 3' non-coding regions diverge both in length and sequence. Within the 5' non-coding region adjacent to the initiating AUG there is a highly conserved 18 bp sequence which is also present in another gene coding for a member of a different, unrelated high-sulphur keratin family. In the B2A-B2D intergene region, tightly linked to the B2D gene, there is a putative, divergently transcribed gene.     

Cloning, mapping, and expression of two novel actin genes, actin-like-7A (ACTL7A) and actin-like-7B (ACTL7B), from the familial dysautonomia candidate region on 9q31.

Two novel human actin-like genes, ACTL7A and ACTL7B, were identified by cDNA selection and direct genomic sequencing from the familial dysautonomia candidate region on 9q31. ACTL7A encodes a 435-amino-acid protein (predicted molecular mass 48.6 kDa) and ACTL7B encodes a 415-amino-acid protein (predicted molecular mass 45. 2 kDa) that show greater than 65% amino acid identity to each other. Genomic analysis revealed ACTL7A and ACTL7B to be intronless genes contained on a common 8-kb HindIII fragment in a "head-to-head" orientation. The murine homologues were cloned and mapped by linkage analysis to mouse chromosome 4 in a region of gene order conserved with human chromosome 9q31. No recombinants were observed between the two genes, indicating a close physical proximity in mouse. ACTL7A is expressed in a wide variety of adult tissues, while the ACTL7B message was detected only in the testis and, to a lesser extent, in the prostate. No coding sequence mutations, genomic rearrangements, or differences in expression were detected for either gene in familial dysautonomia patients.     

The complete map of the Ig heavy chain constant gene region reveals evidence for seven IgG isotypes and for IgD in the horse

This report contains the first map of the complete Ig H chain constant (IGHC) gene region of the horse (Equus caballus), represented by 34 overlapping clones from a new bacterial artificial chromosome library. The different bacterial artificial chromosome inserts containing IGHC genes were identified and arranged by hybridization using overgo probes specific for individual equine IGHC genes. The analysis of these IGHC clones identified two previously undetected IGHC genes of the horse. The newly found IGHG7 gene, which has a high homology to the equine IGHG4 gene, is located between the IGHG3 and IGHG4 genes. The high degree of conservation shared between the nucleotide sequences of the IGHG7 and IGHG4 genes is unusual for the IGHG genes of the horse and suggests that these two genes duplicated most recently during evolution of the equine IGHG genes. Second, we present the genomic nucleotide sequence of the equine IGHD gene, which is located downstream of the IGHM gene. Both the IGHG7 and IGHD genes were found to be expressed at the mRNA level. The order of the 11 IGHC genes in the IGH-locus of the horse was determined to be 5'-M-D-G1-G2-G3-G7-G4-G6-G5-E-A-3', confirming previous studies using lambda phage clones, with the exception that the IGHG5 gene was found to be the most downstream-located IGHG gene. Fluorescence in situ hybridization was used to localize the IGHC region to Equus caballus (ECA) 24qter, the horse chromosome corresponding to human chromosome 14, where the human IGH locus is found.     

Structure of two human beta-actin-related processed genes one of which is located next to a simple repetitive sequence.

From a human gene library we have isolated and sequenced a beta-actin-like pseudogene, H beta Ac-psi 2, which lacks intervening sequences and contains several mutations resulting in frame-shifts, stop codons and in a departure from the known beta-actin protein sequence. We have also extended our sequence work on the intronless human beta-actin-related pseudogene H beta Ac-psi 1 described previously and we find that both genes are processed genes ending in a poly(dA) tract and flanked by direct repeats. The gene H beta Ac-psi 2 is preceded by a 230-bp region in which the simple sequence 5'-GAAA-3' is repeated greater than 40 times. This satellite-like sequence is highly repetitive in the human genome.     

An expressed beta-tubulin gene, TUBB, is located on the short arm of human chromosome 6 and two related sequences are dispersed on chromosomes 8 and 13

The chromosomal assignments of an expressed beta-tubulin gene and two related sequences have been determined by Southern blot analysis of DNA from a panel of human x Chinese hamster somatic cell hybrids cleaved with Hind III or EcoRI. Probes containing the 3' untranslated regions of the expressed gene M40 and of pseudogene 21 beta were used to localize the M40 sequence (gene symbol TUBB) to chromosome 6 region 6p21----6pter, the 21 beta pseudogene (TUBBP1) to chromosome 8 region 8q21----8pter and a third related sequence (TUBBP2) to chromosome 13. Asynteny of expressed genes and related processed pseudogenes has now been demonstrated for several gene families.     

Characterization and Expression of the Human A2a Adenosine Receptor Gene

Abstract: The actions of the neurotransmitter adenosine are mediated by a family of high-affinity, G protein-coupled receptors. We have characterized the gene for the human A2a subtype of adenosine receptor (hA2aR) and determined levels of A2aR mRNA in human brain regions and nonneural tissues. Human genomic Southern blot analysis demonstrates the presence of a single gene encoding the hA2aR located on chromosome 22. Two overlapping cosmids containing the hA2aR gene were isolated from a chromosome 22 library and characterized. Southern blot and sequence analyses demonstrate that the hA2aR gene spans ∼9–10 kb with a single intron interrupting the coding sequence between the regions encoding transmembrane domains III and IV. The sequence of the hA2aR gene diverged from the reported cDNA structure in the 5' untranslated region. This divergence appears to result from an artifact in the construction of the original cDNA library. By northern blot analysis, high expression of the hA2aR gene was identified in the caudate nucleus with low levels of expression in other brain regions. High expression was also seen in immune tissues; lesser A2aR expression was detected in heart and lung. The gene for the A2a subtype of receptor for the neurotransmitter adenosine falls in the class of intron containing G protein-coupled receptor genes. Expression in the basal ganglia is consistent with a role for the hA2aR in motor control. Activation of the A2aR may also regulate immune responses and cardiopulmonary function.     

Structure and chromosome localization of the human eosinophil-derived neurotoxin and eosinophil cationic protein genes: Evidence for intronless coding sequences in the ribonuclease gene superfamily

Human genomic DNAs for the eosinophil granule proteins, eosinophil-derived neurotoxin (EDN) and eosinophil cationic protein (ECP), were isolated from genomic libraries. Alignment of EDN (RNS2) and ECP (RNS3) gene sequences demonstrated remarkable nucleotide similarities in noncoding sequences, introns, and flanking regions, as well as in the previously known coding regions. Detailed examination of the 5'-noncoding regions yielded putative TATA and CAAT boxes, as well as similarities to promoter motifs from unrelated genes. A single intron of 230 bases was found in the 5' untranslated region and we suggest that a single intron in this region and an intronless coding region are features common to many members of the RNase gene superfamily. The RNS2 and RNS3 genes were localized to the q24-q31 region of human chromosome 14. It is likely that these two genes arose as a consequence of a gene duplication event that took place approximately 25-40 million years ago and that a subset of anthropoid primates possess both of these genes or closely related genes.     

Structure, chromosome location, and expression of the human smooth muscle (enteric type) gamma-actin gene: evolution of six human actin genes.

Recombinant phages that carry the human smooth muscle (enteric type) gamma-actin gene were isolated from human genomic DNA libraries. The amino acid sequence deduced from the nucleotide sequence matches those of cDNAs but differs from the protein sequence previously reported at one amino acid position, codon 359. The gene containing one 5' untranslated exon and eight coding exons extends for 27 kb on human chromosome 2. The intron between codons 84 and 85 (site 3) is unique to the two smooth muscle actin genes. In the 5' flanking region, there are several CArG boxes and E boxes, which are regulatory elements in some muscle-specific genes. Hybridization with the 3' untranslated region, which is specific for the human smooth muscle gamma-actin gene, suggests the single gene in the human genome and specific expressions in enteric and aortic tissues. From characterized molecular structures of the six human actin isoform genes, we propose a hypothesis of evolutionary pathway of the actin gene family. A presumed ancestral actin gene had introns at least sites 1, 2, and 4 through 8. Cytoplasmic actin genes may have directly evolved from it through loss of introns at sites 5 and 6. However, through duplication of the ancestral actin gene with substitutions of many amino acids, a prototype of muscle actin genes had been created. Subsequently, striated muscle actin and smooth muscle actin genes may have evolved from this prototype by loss of an intron at site 4 and acquisition of a new intron at site 3, respectively.