Rabbit Genomic DNA Research Articles

Structure and functional regulation of the CD38 promoter

CD38 has multiple roles in biology, including T lymphocyte signaling, neutrophil migration, neurotransmission, cell proliferation, apoptosis, and bone remodeling. To study the transcriptional control of the CD38 gene, we cloned a putative 1.8 kb promoter fragment from a rabbit genomic DNA library. Primer extension analysis indicated two transcription start sites consistent with the absence of a TATA box. Sequence analysis revealed several AP-1, AP-4, myo-D, GATA, and SP-1 sequences. MC3T3.E1 (osteoblast) or RAW-C3 (osteoclast precursor macrophage) cells were then transfected with the CD38 promoter or its deletion fragments ligated to the luciferase reporter gene. Except for the shortest 41 bp fragment, all fragments showed significant luciferase activity. There was a marked stimulation of basal activity in the 93 bp fragment that contained a GC box and SP-1 site. Furthermore, there were significant differences in the activity of the fragments in MC3T3.E1 and RAW-C3 cells. Intracellular Ca 2+ elevations by ionomycin (10 μM) in MC3T3.E1 cells inhibited promoter activity, except in the short 41 bp. In contrast, cAMP elevation by exposure to forskolin (100 μM) inhibited activation of all fragments, except the 0.6 and 1.2 kb fragments. Finally, TNF-α stimulated promoter activity in RAW-C3 cells transfected with the 93 bp and 1.0 kb fragments, consistent with the stimulation of CD38 mRNA by TNF-α. Physiologically, therefore, modulation of the expression of the NAD +-sensing enzyme, CD38, by Ca 2+, cAMP, and cytokines, such as TNF-α may contribute to coupling the intense metabolic activity of osteoclasts and osteoblasts to their respective bone-resorbing and bone-forming functions.

Rabbit EPO Gene and cDNA: Expression of Rabbit EPO after Intramuscular Injection of pDNA

Erythropoietin (EPO) cDNA was cloned from kidney total RNA of a NZW rabbit. The cDNA comprises a 588-bp open reading frame encoding a 195 amino acid protein with distinguishable regions of high of homology to other mammalian EPOs. Intramuscular injection of mice with a rabbit EPO expression plasmid resulted in a significant hematocrit increase. A rabbit genomic DNA fragment was also cloned using the rabbit EPO cDNA. This 4312-bp genomic DNA fragment contains sequences homologous to the mouse EPO promoter and hypoxia-responsive enhancer. In addition, the genomic DNA also presents a high degree of conservation to other regions involved in hypoxia response. Sequence divergence in the 3′ UTR may indicate differences in regulation of mRNA stability or response to low oxygen tension.

5′-Flanking Sequence and Promoter Activity of the Rabbit Neuronal Nitric Oxide Synthase (nNOS) Gene

We have isolated a rabbit neuronal nitric oxide synthase (nNOS) cDNA encoding a protein of 1,435 amino acids. Using the cDNA clones as probes, the 5′-flanking region of the nNOS gene was isolated from a rabbit genomic DNA library. 5′RACE and primer extension analysis of rabbit brain total RNA mapped multiple transcription initiation sites localized 474-487 bp upstream from the translation start codon. Analysis of 5,197 bp of the 5′-flanking sequence revealed that the rabbit nNOS gene promoter lacks canonical TATA or CCAAT boxes and, instead, contains a GC-rich region and multiple Sp1 sites. Farther from the + 1 start, various putative cis-elements including AP-1, AP-4, NF-KB, STAT, CREB, C/EBP and c-Myc were observed. The functional promoter activity of the 5′-flanking region was demonstrated by its ability to drive the expression of a β-galactosidase reporter gene in several cell types. Serial deletion analysis of the promoter region revealed that the -291 to -172 region, which contains two Sp1 sites, is essential for basal transcriptional activity. These results suggest that the rabbit nNOS promoter contains characteristics of inducible genes.

5'-Flanking sequence and promoter activity of the rabbit neuronal nitric oxide synthase (nNOS) gene.

We have isolated a rabbit neuronal nitric oxide synthase (nNOS) cDNA encoding a protein of 1,435 amino acids. Using the cDNA clones as probes, the 5'-flanking region of the nNOS gene was isolated from a rabbit genomic DNA library. 5'RACE and primer extension analysis of rabbit brain total RNA mapped multiple transcription initiation sites localized 474-487 bp upstream from the translation start codon. Analysis of 5,197 bp of the 5'-flanking sequence revealed that the rabbit nNOS gene promoter lacks canonical TATA or CCAAT boxes and, instead, contains a GC-rich region and multiple Sp1 sites. Farther from the +1start, various putative cis-elements including AP-1, AP-4, NF-kappaB, STAT, CREB, C/EBP and c-Myc were observed. The functional promoter activity of the 5'-flanking region was demonstrated by its ability to drive the expression of a beta-galactosidase reporter gene in several cell types. Serial deletion analysis of the promoter region revealed that the -291 to -172 region, which contains two Sp1 sites, is essential for basal transcriptional activity. These results suggest that the rabbit nNOS promoter contains characteristics of inducible genes.

Complementary deoxyribonucleic acid cloning and tissue expression of BSP-A3 and BSP-30-kDa: phosphatidylcholine and heparin-binding proteins of bovine seminal plasma.

BSP-A1, BSP-A2, BSP-A3, and BSP-30-kDa are four major proteins of bovine seminal plasma (BSP protein family). These heparin- and phosphatidylcholine-binding proteins potentiate the capacitation of spermatozoa. Here we determined the complete sequences of the two cDNAs coding for the BSP-A3 and BSP-30-kDa proteins. Degenerate oligonucleotides designed on the basis of the primary sequences of the proteins were used as primers in reverse transcription-polymerase chain reaction, with cDNA preparations of bovine seminal vesicles as templates, to amplify an internal fragment of each BSP cDNA. Specific oligonucleotides designed on the basis of these partial cDNA sequences were used to clone the two complete cDNAs by using the 3' rapid amplification of cDNA ends (RACE) and 5' RACE methods. We also verified the expression of all members of the bovine BSP protein family in several adult bovine tissues by RNase protection assays. The results indicated that each BSP protein mRNA is expressed only in seminal vesicles and in the ampullae. Homologous genes were detected in human, rat, hamster, and rabbit genomic DNA, using high-stringency Southern hybridization with a specific BSP-30-kDa cDNA probe.

Cloning of Rat Muc5AC Mucin Gene: Comparison of Its Structure and Tissue Distribution to That of Human and Mouse Homologues

The human mucin gene MUC5AC codes for a large mucin which has tandem repeat units and cysteine rich regions characteristic of several members of this class of glycoproteins. Human epithelia expressing the mucin include that of stomach, bronchus/trachea, endocervix and conjunctiva. We report here a 3.8 kb partial sequence of a rat homologue for the human MUC5AC gene and compare its tandem repeat sequence and cysteine rich domains to those of the human and mouse gene. Rat and mouse have the same number of amino acids (16) in their Muc5AC tandem repeat units and share 69% sequence similarity, whereas human MUC5AC has only 8 amino acids in its tandem repeat. In rat, the tandem repeat domain is flanked at its 3′ end by a non-repeat region coding for 1142 amino acids. Four cysteine rich subdomains were identified in this region; one of these has 64% similarity to a corresponding region in human MUC5AC and 80% similarity to a mouse MUC5AC cysteine rich region. Southern blot analysis revealed cross hybridization of a probe for the rat cysteine rich region, to human, mouse, rabbit, and porcine genomic DNA; the rat tandem repeat probe hybridized with mouse and rabbit only. Unlike humans, rat expressed MUC5AC message detectable by Northern blot andin situhybridization only in stomach epithelium and conjunctival goblet cells.

Macrophage TNF mRNA Expression Is Modulated by Protease Inhibitors

Background: Nuclear factor κB (NFκB) is an important transcriptional activator protein and is a crucial component of the host's response to infection. The activation of NFκB is correlated with the phosphorylation of inhibitory κB (IκB) and its subsequent degradation. We hypothesized that protease inhibitors which prevented IκB degradation could inhibit the macrophage gene activation and reduce the production of inflammatory cytokines. Methods: Rabbit alveolar macrophages (Mφ) were obtained by bronchoalveolar lavage. Mφ were exposed toEscherichia colilipopolysaccharide (LPS) (10 ng/ml) in the presence of various concentrations of protease inhibitors, eitherN-tosyl-l-phenylalanine chloromethyl ketone (TPCK) orN-benzoyl-l-tyrosine ethyl ester (BTEE). Total RNA was extracted for Northern blot assay of tumor necrosis factor (TNF) mRNA expression using a rabbit genomic DNA probe. Total nuclear extracts were also obtained for the measurement of the NFκB activity with the electrophoretic mobility shift assay. The TNF production in the Mφ supernatant was measured by L929 bioassays. Results: NFκB activity induced by LPS was inhibited by either BTEE or TPCK. Inhibition of NFκB activity by these agents also prevented TNF mRNA expression and TNF production induced by LPS. The cellular mechanism leading to NFκB activation was further studied. TNF mRNA expression and NFκB activation were inhibited by D609, a phospholipase C (PLC) inhibitor, as well as by protein kinase C (PKC) inhibitors. In addition, direct stimulation of PKC led to NFκB activation and TNF mRNA expression. Conclusions: These data suggest that TNF mRNA expression of LPS-stimulated Mφ is mediated through NFκB. NFκB activation is intimately regulated by the PLC signaling pathway.

CHARACTERIZATION OF A FULL-LENGTH cDNA FOR RABBIT FACTOR X

A 300 bp probe generated by the PCR was derived from rabbit genomic DNA using primers from a highly conserved region of the DNA for human factor X (HFX). The probe was used in northern blot analysis of liver RNA to demonstrate an mRNA species of 1.6 kb for the rabbit factor X and subsequently for isolation and characterization of the cDNA for rabbit factor X (RFX) from a lambda Zap II cDNA library generated from rabbit liver mRNA. The cDNA contains 22 bases upstream from the 5′-translation initiation codon, 1470 nucleotides of open reading frame, a stop codon and a 3′ poly (A) tail. The cDNA codes for a 40-residue signal/propeptide region, followed by a 447-residue mature protein. The deduced amino acid sequence shows a high degree of homology with the sequence of HFX. Inhibitory peptides derived from interactive sites of HFX for activators, cofactor and substrate exerted degrees of inhibition of RFX activation which showed a dependence on extent of homology with the corresponding regions of RFX. © 1997 Elsevier Science Ltd

Reinterpretation of the RACTK1 K+ channel.

The RACTK1 cDNA cloned from rabbit kidney cortical collecting duct cells was associated with inwardly rectifying pH-regulated K+ channel activity (M. Suzuki, K. Takahashi, M. [keda, H Hayakawa, A. Ogawa, Y. Kawaguchi, and O. Sakai. Nature Lond. 367: 642-645, 1994). The deduced amino acid sequence of the encoded novel polypeptide lacked the signature sequence of a K(+)-selective pore region but predicted a topography suggestive of the inward rectifier K+ channel family. In subsequent articles a RACTK1 epitope was immunolocalized to the apical surface of kidney collecting duct and to arteriolar smooth muscle [M. Suzuki, T. Takigawa, K. Kimura, C. Koseki, and M. Imai. Am. J. Physiol. 269 (Cell Physiol, 38): C496-C503, 1995], and apamin-sensitive K+ currents displaying Ca(2+)-dependent and voltage-independent activation accompanied stable heterologous overexpression of RACTK1 [M. Suzuki, M. Murata, M. Ikeda, T. Miyoshi, and M. Imai. Am. J. Physiol. 270 (Cell Physiol, 39): C964-C968, 1996]. We now report that the "RACTK1" open reading frame is a frame-shifted translation of the antisense strand of an Escherichia coli gene member of a coenzyme A transferase gene family. "RACTK1" mRNA was absent from tissues free of E. coli contamination, and the "RACTK1" gene was undetectable in Southern blots of human and rabbit genomic DNA. We conclude that the immunostaining patterns and Ca(2+)-activated K+ channel activity heretofore attributed to RACTK1 must be otherwise explained.

Molecular Cloning and Expression of a Third Type of Rabbit GDP-L-Fucose:β-D-Galactoside 2-α-L-Fucosyltransferase

Recent molecular investigation revealed that two closely related structural genes encode distinct GDP-L-fucose:beta-D-galactoside 2-alpha-L-fucosyltransferases (alpha1,2-fucosyltransferases). Some human cancer cells or tissues may express an aberrant alpha1, 2-fucosyltransferase other than H- and Secretor-type alpha1, 2-fucosyltransferase. However, definite evidence of the existence of a third type of alpha1,2-fucosyltransferase has not been demonstrated. Here we report the molecular cloning of a third type of rabbit alpha1,2-fucosyltransferase (RFT-III) from a rabbit genomic DNA library. The DNA sequence included an open reading frame coding for 347 amino acids, and the deduced amino acid sequence of RFT-III showed 59 and 80% identity with those of the previously reported two types of rabbit alpha1,2-fucosyltransferase, RFT-I and RFT-II, respectively. COS-7 cells transfected with the RFT-III gene exhibited alpha1,2-fucosyltransferase activity toward phenyl-beta-Gal as a substrate. Neuro2a (a murine neuroblastoma cell line) cells transfected with the RFT-III gene expressed fucosyl GM1 (type 3 H) but not Ulex europaeus agglutinin-1 lectin reactive antigens (type 2 H). Kinetic studies revealed that RFT-III exhibits higher affinity to types 1 (Galbeta1, 3GlcNAc) and 3 (Galbeta1, 3GalNAc) than to type 2 (Galbeta1, 4GlcNAc) oligosaccharides, which suggests that RFT-III as well as RFT-II is a Secretor-type alpha1, 2-fucosyltransferase. RFT-III was expressed in the adult gastrointestinal tract. The RFT-I, -II, and -III genes were assigned within 90 kilobases on pulsed field gel electrophoresis analysis. These results constitute direct evidence that, at least in one mammalian species, three active alpha1,2-fucosyltransferases exist.

Molecular Cloning and Expression of Two Types of Rabbit β-Galactoside α1,2-Fucosyltransferase

Two DNA clones encoding rabbit beta-galactoside alpha 1,2-fucosyltransferase (RFT-I and RFT-II) have been isolated from a rabbit genomic DNA library. The DNA sequences revealed open reading frames coding for 373 (RFT-I) and 354 (RFT-II) amino acids, respectively. The deduced amino acid sequences of RFT-I and RFT-II showed 56% identity with each other, and that of RFT-I showed 80% identity with that of human H blood type alpha 1,2-fucosyltransferase. Northern blot analysis of embryo and adult rabbit tissues revealed that the RFT-I gene was expressed in adult brain, and that the RFT-II gene was expressed in salivary and lactating mammary glands. The identities of these enzymes were confirmed by constructing recombinant fucosyltransferases in which the N-terminal part including the cytoplasmic tail and signal anchor domain was replaced with the immunoglobulin signal peptide sequence. RFT-I expressed in COS-7 cells exhibited similar transferase activity to that of human H blood type alpha 1,2-fucosyltransferase. RFT-II expressed in COS-7 cells showed higher affinity for type 1 (Gal beta 1,3GlcNAc) and type 3 (Gal beta 1,3GalNAc) acceptors than type 2 (Gal beta 1,4GlcNAc) ones, which suggested that RFT-II was a putative secretor-type alpha 1,2-fucosyltransferase.

QC-PCR accurate determination of rabbit genomic DNA: Evaluation of VZV load in the rabbit ocular infection model: Q Zhu 1, M Piatak 2, J Guo 1, S Yamamoto, 1 D Pavan-Langston, 1 and EC Dunkel 1. 1Schepens Eye Research Institute; Department of Ophthalmology, Harvard Medical School, Boston, MA and 2Gene Labs, Inc. Redwood City, CA, USA

QC-PCR accurate determination of rabbit genomic DNA: Evaluation of VZV load in the rabbit ocular infection model: Q Zhu 1, M Piatak 2, J Guo 1, S Yamamoto, 1 D Pavan-Langston, 1 and EC Dunkel 1. 1Schepens Eye Research Institute; Department of Ophthalmology, Harvard Medical School, Boston, MA and 2Gene Labs, Inc. Redwood City, CA, USA

Human homolog of a mouse sequence from the dystonia musculorum locus is on Chromosome 6p12

Dystonia musculorum is a hereditary neurodegenerative disease in mice that affects sensory neurons. In an effort to clone the gene responsible for this disorder, we have assembled a genomic contig spanning 75 kb of the dystonia musculorum (dt) locus. Within this genomic contig, we have identified a small restriction fragment that shows evolutionary conservation to rat, hamster, rabbit, and human genomic DNA. Using this mouse sequence, we have cloned the conserved human genomic fragment. Sequence analysis of the mouse and human genomic fragments revealed that they share a sequence similarity of 82% over 175 bp. A panel of human/rodent somatic cell hybrids was used to map the human genomic sequence to Chromosome (Chr) 6, and high-resolution in situ hybridization (FISH) allowed it to be sublocalized to 6p12. The human homolog of the mouse Bpag1 gene, a gene tightly linked to the mouse dt gene, also maps to Chr 6. Thus, this comparative mapping reveals a new region of conserved synteny between the chromosomes of mouse and human. Mapping the human homolog of the mouse dt gene enables us to initiate linkage studies to identify neurodegenerative disorders that may be caused by mutations in this gene.

Cloning and sequencing of flavin-containing monooxygenases FMO3 and FMO4 from rabbit and characterization of FMO3.

The flavin-containing monooxygenases (FMO) are a family of enzymes that contain putative FAD- and NADPH-binding domains within the first 200 residues of their N termini. The cDNAs encoding these enzymes contain an area of relatively high identity over the 5' half of the coding region. Rabbit genomic DNA was probed under low stringency conditions, with a mixture of 5' cDNA fragments encoding rabbit FMO1, FMO2, or FMO5. Bands associated specifically with FMO1, FMO2, or FMO5 were resolved by analysis at high stringency with individual probes. Several bands were detected that could not be assigned to FMO1, FMO2, or FMO5. The behavior of the 5' probes at low versus high stringency was used to facilitate the isolation of cDNAs corresponding to the unknown DNA bands. A cDNA library was constructed from rabbit liver mRNA and screened under low stringency hybridization conditions (30 degrees C, 50% formamide, 1 x SSC, 0.1% SDS) with the mixture of 5' FMO1, FMO2, and FMO5 cDNA probes. A total of 157 clones was detected. Of these, 117 clones remained under high stringency hybridization conditions (65 degrees C, 50% formamide, 0.1 x SSC, 0.1% SDS) and were identified as FMO1 (95 clones) or FMO5 (22 clones). Of the 40 remaining clones, 36 were characterized by sequence analysis as encoding FMO3, previously identified at the protein level by Ozols (Ozols, J. (1991) Arch. Biochem. Biophys. 290, 103-115) as a second rabbit liver FMO. Four clones were shown to encode an FMO not previously described for the rabbit, FMO4. No clones encoding FMO2 were isolated from the liver library. Sequence analysis revealed that FMO3 and FMO4 are 56% identical, and analysis of genomic DNA indicated that each is encoded by a single gene. Message distribution was tissue-, species-, and form-specific. The properties of FMO3 cDNA expressed in Escherichia coli were found to be more similar to those of FMO1 than FMO2, but to differ significantly from both. Rabbit genomic DNA was probed under conditions of low stringency with a mixture of 5' cDNA fragments encoding all five FMO forms and produced results consistent with the possibility of one additional FMO.

Alternative splicing generates multiple isoforms of a rabbit prostaglandin E2 receptor.

Four cDNA clones homologous with a murine prostaglandin E2 receptor have been isolated from a rabbit kidney cortex cDNA library. These cDNAs encode related proteins that differ only in their COOH-terminal sequences. Southern blot analysis of rabbit genomic DNA indicates that these receptor cDNAs represent alternatively spliced variants derived from a single gene. This was confirmed by isolation and sequence analysis of genomic clones containing common region exons and unique 3'-coding exons, which contained intron/exon boundaries at the predicted splice junctions. Transient expression of a novel full-length cDNA in COS1 cells confirmed the ligand binding profile typical of an EP3 receptor subtype. Ribonuclease protection assays indicate that the gene encoding these receptors is most highly expressed in kidney, adrenal, and stomach with lower but significant expression in uterus, lung, heart, ileum, spleen, and brain. Moreover, each of the cloned isoforms is expressed in the kidney. In situ hybridization analyses of rabbit kidney demonstrated that the level of expression is highest in the outer medulla with lesser expression in the cortex and no detectable expression in the inner medulla. The ligand binding profile and tissue distribution of these receptors is consistent with a functional role for this family of EP3 receptors in mediating the renal actions of prostaglandins as well as the effects of prostaglandins on gastric acid secretion and adrenal function.

Cloning, sequencing, distribution, and expression in Escherichia coli of flavin-containing monooxygenase 1C1. Evidence for a third gene subfamily in rabbits

Two full-length cDNA clones (2.2 kilobases) encoding a newly recognized form of mammalian flavin-containing monooxygenase (FMO) have been isolated from independent libraries constructed with mRNA from different rabbits. The cDNAs encode a polypeptide of 533 amino acids which contains two putative pyrophosphate binding domains and a hydrophobic carboxyl terminus characteristic of FMOs. This sequence is 52 and 57% identical to sequences of the rabbit "hepatic" and "pulmonary" FMOs, respectively, and 55% identical to the sequence of "liver form 2" published recently by Ozols (Ozols, J. (1991) Arch. Biochem. Biophys. 290, 103-115). cDNA for the new FMO (FMO 1C1) hybridizes with two species of mRNA, one of 2.6 kilobases and one of about 5.4 kilobases, from liver or kidney, but not lung. Guinea pig, hamster, rat, and mouse all express this form of FMO in liver, kidney, and lung. FMO 1C1 has been tentatively characterized following expression in Escherichia coli. It is inactive with methimazole as substrate but highly active with n-octylamine. The temperature lability, responses to ions and detergent, and pH optimum of FMO 1C1 are similar to values reported for hepatic FMO. Sequence comparisons and analysis of rabbit and human genomic DNA indicate that FMO 1C1, as well as the pulmonary and hepatic FMOs, comprise a single gene family made up of distinct gene subfamilies (A, B,C,D, ... N), each appearing to contain a single gene. A nomenclature, based on these interrelationships and following the same designations used for classifying cytochromes P-450, is proposed.

Cloning of a rabbit kidney cortex AT1 angiotensin II receptor that is present in proximal tubule epithelium

The rabbit proximal tubule (PT) has been widely utilized to study the direct effects of angiotensin II (ANG II) on PT function. The purpose of the present study was to characterize the binding properties of PT ANG II receptors, using nonpeptide antagonists, and to clone a rabbit PT ANG II receptor. In rat and rabbit kidney cortical brush-border and basolateral membranes, specific binding of 125I-ANG II was inhibited by the AT1 ANG II-receptor antagonist DuP 753, but not by the AT2 antagonist PD 123319. Using a rabbit kidney cortex cDNA library, we isolated cDNA encoding an ANG II receptor, with an open-reading frame sharing a high degree of sequence homology to previously cloned AT1 ANG II receptors. In transfected COS-1 cells, this rabbit ANG II receptor had properties of the AT1 class. Northern analysis revealed high levels of mRNA expression for this receptor in rabbit kidney cortex and adrenal gland. Within the kidney, message was detected in primary cultures of rabbit PT cells, as well as in freshly isolated rabbit PT segments. Message was also present in cells of the mouse PT line, MCT, and in rat glomerular mesangial cells. Utilizing polymerase chain reaction (PCR) with primers derived from the 1st and 4th transmembrane domains of the rat AT1A ANG II receptor, a 279-bp DNA fragment was amplified from reverse-transcribed RNA from rabbit PT cells. This DNA encoded an amino acid sequence identical to that encoded by the rabbit kidney cDNA clone in the corresponding region and differed by a single base substitution. Southern analysis of rabbit genomic DNA restriction digests with the rabbit ANG II receptor probe revealed hybridization to a single band in each lane. These results indicate that an AT1 ANG II receptor is present in the PT and that a single gene codes for the AT1 receptor in rabbit. The clone isolated in the present study should provide a useful tool with which to study the regulation of the PT renin-angiotensin system.

Cloning and structural characterization of a rabbit genomic DNA for α1 acid glycoprotein

The gene for rabbit alpha 1 acid glycoprotein (AGP) has been isolated from a lambda EMBL3 genomic DNA library. Isolated clone contains a 12 Kbp fragment of rabbit genomic DNA. Restriction endonuclease mapping has localized the gene within a 4.2 Kbp fragment spanning two EcoRI sites. Southern blot analysis of the rabbit genomic DNA and its comparison with the cloned gene indicates that there is only one gene for AGP present per genome. DNA sequence analysis of the cloned gene indicates that the entire gene, TATA box to the polyadenylation signal, is located within the 4.2 Kbp region and contains six exons representing the full-length cDNA described earlier (1). The 5'-end of alpha 1-AGP gene sequences from rabbit, human, rat and mouse have been compared. Such analysis reveals two conserved regions located between -63 bp and -36 bp and -29 bp and -1 bp of putative transcription start site, which may play a role in transcriptional induction of this gene during acute response. In addition to this conserved domain, DNA sequence upstream of the major transcription start site contains a potential element for Sp1 binding and a 18 bp long palindrome sequence followed by a short repeating dinucleotide sequence, which may be important in the regulation of AGP gene induction.

Isolation of putative voltage-gated epithelial K-channel isoforms from rabbit kidney and LLC-PK1 cells

Epithelial voltage-gated potassium (K) channels have been well studied using electrophysiological methods, but little is known about their structures. We tested the hypothesis that some of these channels belong to the Shaker gene family, which encodes voltage-gated K channels in excitable tissues. From published sequences of Shaker proteins in Drosophila, rat, and mouse brain, we chose regions that were conserved between species. Based on these protein sequences, degenerate oligonucleotides flanking the putative voltage sensor (S4) were synthesized and used as primers for the polymerase chain reaction. Five Shaker-like cDNAs were amplified from rabbit kidney cortex and three from LLC-PK1, an epithelial cell line derived from pig kidney. Each partial-length rabbit kidney cDNA is approximately 850 base pairs (bp) long. The deduced amino acid sequences contain five putative transmembrane segments and are 79-97% identical to two Shaker isoforms expressed in rat brain (RBK1 and RBK2). Sequence similarity is greatest in the putative transmembrane segments S1-S5. Importantly, the S4 segment, the putative voltage gate is highly conserved in all 5 cDNAs. Southern analysis of rabbit genomic DNA suggests that each isoform is encoded by a different gene. The partial length LLC-PK1 cDNAs are 450-bp long, and the deduced amino acid sequences are 77-99% identical to the rabbit cDNAs. This is, to our knowledge, the first demonstration that Shaker-like genes are expressed in renal epithelial cells. These genes most likely encode voltage-gated K channels involved in renal epithelial K transport.

Molecular characterization of the interleukin-8 receptor

Recently a rabbit cDNA (F3R) was characterized as binding and causing calcium mobilization induced by the formyl-methionine-leucine-phenylalanine peptide (fMLP). In the study reported here, cloned DNAs were isolated from rabbit genomic DNA by PCR based on the sequence of F3R. The cloned DNAs have several differences in the DNA sequence compared to the reported F3R sequence that alter the predicted protein sequence. COS-7 cells transfected with these clones in a mammalian expression vector bind human IL-8 with high affinity, but do not bind fMLP. We therefore believe that the cDNAs isolated encode the rabbit IL-8 receptor.