Rapid Amplification Of cDNA Ends Method Research Articles

Cloning and expression of the bovine 11β-hydroxysteroid dehydrogenase type-2

The bovine 11β-hydroxysteroid dehydrogenase type 2 enzyme (11β-HSD-2) cDNA was cloned from three overlapping PCR fragments using primers based on the human and ovine 11β-HSD-2 cDNA sequences. Both cDNA ends were obtained by a modified RACE (Rapid Amplification of cDNA Ends) method. The bovine 11β-HSD-2 cDNA is 1878 bp long, excluding the poly(A) tail. It consists of a 5′-untranslated region of 133 bp, an open reading frame of 1215 bp and a 3′-untranslated region of 530 bp. Bovine 11β-HSD-2 cDNA is highly homologous to that of the sheep (92%) and less related to the human (67%), rabbit (65%), rat (52%) and mouse (45%) cDNA. The predicted bovine 11β-HSD-2 protein contains 404 amino acid residues with a calculated mol wt of 43,985. It is homologous to the sheep (98%) and human (88%) protein, and less related to that of the rabbit (76%), rat (80%) and mouse (77%). The cloned 11β-HSD-2 cDNA was transfected into CHOP cells and the enzymatic characteristics determined. The enzyme functions primarily as an oxidase, uses NAD + and is more active with corticosterone as a substrate than with cortisol or dexamethasone. It is expressed in high concentrations in kidney, adrenal and colon, and in small concentrations in liver, heart and lung. In conclusion, the 11β-HSD-2 enzyme of cattle is very similar to that of other species in its structure and enzymatic characteristics.

Optimized rapid amplification of cDNA ends (RACE) for mapping bacterial mRNA transcripts.

A simple, efficient and sensitive RACE-based procedure was developed for the determination of unknown 5' regions from bacterial cDNA. A number of critical modifications were made to the standard RACE method, including the optimization of the RNA extraction, reverse transcription and PCR conditions. This procedure was used to accurately determine the site of transcript initiation and structure of the promoter region of the Helicobacter pylori aspartate carbamoyltransferase gene (pyrB). The technique avoids many of the difficulties associated with established bacterial transcript mapping protocols and can be performed in two days starting with less than 1 microgram of total RNA. The modifications described here have significant potential for the identification of transcript start sites of bacterial genes and non-polyadenylated eukaryotic RNA.

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.

Calumenin, a Ca2+-binding protein retained in the endoplasmic reticulum with a novel carboxyl-terminal sequence, HDEF.

We have identified and characterized a cDNA encoding a novel Ca2+-binding protein named calumenin from mouse heart by the signal sequence trap method. The deduced amino acid sequence (315 residues) of calumenin contains an amino-terminal signal sequence and six Ca2+-binding (EF-hand) motifs and shows homology with reticulocalbin, Erc-55, and Cab45. These proteins seem to form a new subset of the EF-hand protein family expressed in the lumen of the endoplasmic reticulum (ER) and Golgi apparatus. Purified calumenin had Ca2+-binding ability. The carboxyl-terminal tetrapeptide His-Asp-Glu-Phe was shown to be responsible for retention of calumenin in ER by the retention assay, immunostaining with a confocal laser microscope, and the deglycosylation assay. This is the first report indicating that the Phe residue is included in the ER retention signal. Calumenin is expressed most strongly in heart of adult and 18.5-day embryos. The calumenin gene (Calu) was mapped at the proximal portion of mouse chromosome 7.

Enhanced RACE method using specific enrichment by biotinylated oligonucleotides bound to streptavidin coated magnetic particles.

RACE (rapid amplification of cDNA ends) is commonly used for identification and isolation of 3'and 5'termini of cDNA. We developed an improvement of the RACE-method that allows the enrichment of wanted fragments. The important new feature is the purification of the amplified products by biotinylated oligonucleotides that hybridize internally. Hybrids are isolated by streptavidin coated magnetic particles.

Three different calmodulin-encoding cDNAs isolated by a modified 5'-RACE using degenerate oligodeoxyribonucleotides

In order to obtain the 5' ends of the three mouse calmodulin (CaM) cDNAs, we modified the standard 5' RACE (rapid amplification of cDNA ends) method to use degenerate synthetic oligodeoxyribonucleotides to prime cDNA synthesis of all three CaM mRNAs. In this modified method, the degenerate primers were annealed to mRNAs in an incubation step prior to the reverse transcription reaction. Separating the annealing step from the reverse transcription reaction allowed for greater stringency by using higher temperatures than could be tolerated if the reverse transcriptase were present. Annealing was also done with lower primer concentration and was driven by a longer incubation time. After the annealing step, cDNA synthesis was initiated by diluting the annealing mixture into a 42°C buffer with reverse transcriptase. The synthesized cDNA was poly (dA) -tailed to allow PCR amplification of the first-strand cDNA with an anchor-dT17 primer and the degenerate primers. The CaM cDNAs were evident after this PCR. A second PCR, with nested gene-specific primers, was used to isolate the individual CaM cDNAs from the products of the first PCR. Three distinct CaM cDNAs were cloned and sequenced. By comparison of the 5' untranslated sequences between the mouse CaM DNAs and rat CaM cDNAs, the corresponding homologs were assigned. The results suggest that application of this modified RACE method could improve the success of isolating specific cDNAs in cases where use of a nested primer is not possible or when amino-acid sequence information is available and only degenerate primers can be designed for cloning cDNAs by the 5'-RACE method.

Cloning, Functional Expression and Tissue Distribution of Human cDNA for the Vascular-Type Vasopressin Receptor

We have cloned a human vasopressin receptor from human mesenteric artery using RACE (Rapid Amplification of cDNA Ends) methods. The deduced amino acid sequence of the clone (HV RACE) encodes a protein of 418 amino acids that showed a strong sequence homology to the previously cloned rat V-1A vasopressin receptor. The [3H] arginine vasopressin (AVP) binding to HV-RACE expressed in COS-7 cells was potently inhibited by AVP (Ki = 2.9 nM). Interestingly, a new non-peptide "V1-selective" antagonist OPC-21268 exhibited markedly higher affinity for rat V1A receptor (Ki = 57 nM) rather than for HV-RACE (Ki = 56 μM). With the reverse-transcription polymerase chain reaction assay, we observed a large amount of HV-RACE transcripts in the mesenteric artery, while a small amount in a variety of other tissues. The data show that the clone HV-RACE encodes a human vascular-type vasopressin receptor cDNA.