Isolation, cloning and expression of a putative abalone gene relating to egg-laying hormone

A putative abalone egg-laying hormone has been amplified by polymerase chain reaction (PCR) from abalone genomic DNA. The PCR product was found to hybridize to Lymnaea stagnalis egg-laying hormone (CDCH) cDNA probe and the PCR product was then cloned and sequenced. Nucleotide sequences of putative abalone egg-laying hormone were determined.

Genomic analyses and cloning of novel chicken natriuretic peptide genes reveal new insights into natriuretic peptide evolution

The natriuretic peptide (NP) family consists of multiple subtypes in teleosts, including atrial, B-type, ventricular, and C-type NPs (ANP, BNP, VNP, CNP-1–4, respectively), but only ANP, BNP, CNP-3, and CNP-4 have been identified in tetrapods. As part of understanding the molecular evolution of NPs in the tetrapod lineage, we identified NP genes in the chicken genome. Previously, only BNP and CNP-3 have been identified in birds, but we characterized two new chicken NP genes by cDNA cloning, synteny and phylogenetic analyses. One gene is an orthologue of CNP-1, which has only ever been reported in teleostei and bichir. The second gene could not be assigned to a particular NP subtype because of high sequence divergence and was named renal NP (RNP) due to its predominant expression in the kidney. CNP-1 mRNA was only detected in brain, while CNP-3 mRNA was expressed in kidney, heart, and brain. In the developing embryo, BNP and RNP transcripts were most abundant 24 h post-fertilization, while CNP mRNA increased in a stage-dependant manner. Synthetic chicken RNP stimulated an increase in cGMP production above basal level in chicken kidney membrane preparations and caused a potent dose-dependant vasodilation of pre-constricted dorsal aortic rings. From conserved chromosomal synteny, we propose that the CNP-4 and ANP genes have been lost in chicken, and that RNP may have evolved from a VNP-like gene. Furthermore, we have demonstrated for the first time that CNP-1 is retained in the tetrapod lineage.

Molecular cloning of natriuretic peptides from the heart of reptiles : loss of ANP in diapsid reptiles and birds

Atrial natriuretic peptide (ANP) and B-type NP (BNP) are hormones involved in homeostatic control of body fluid and cardiovascular regulation. Both ANP and BNP have been cloned from the heart of mammals, amphibians, and teleost fishes, while an additional cardiac peptide, ventricular NP, has been found in selected species of teleost fish. However, in chicken, BNP is the primary cardiac peptide identified thus far. In contrast, the types of NP/s present in the reptilian heart are unknown, representing a considerable gap in our understanding of NP evolution. In the present study, we cloned and sequenced a BNP cDNA from the atria of representative species of reptile, including crocodile, lizard, snake, and tortoise. In addition, we cloned BNP from the pigeon atria. The reptilian and pigeon BNP cDNAs had ATTTA repeats in the 3′ untranslated region, as observed in all vertebrate BNP mRNAs. A high sequence homology was evident when comparing reptile and pigeon preproBNP with the previously identified chicken preproBNP. In particular, the predicted mature BNP-29 was identical between crocodile, tortoise, and chicken, with pigeon having a single amino acid substitution; lizard and snake BNP had seven and nine substitutions, respectively. Furthermore, an ANP cDNA could only be cloned from the tortoise atria. Since ANP was not isolated from the heart of any non-chelonian reptile and appears to be absent in birds, we propose that the ANP gene has been lost after branching of the turtles in the amniote line. This data provides new avenues for research on NP function in reptiles.

Cloning and characterisation of the hint homologue of the Thermophile Thermus thermophilus

Screening of a genomic library of the thermophile Thermus thermophilus revealed a novel thermophilic hint gene, homologues of which are highly conserved in genera from archaea to mammals. Hint belongs to the HIT protein super-family, which contains two broad groups, Fhit, associated with tumour suppression in eukaryotes and Hint with putatitive protein kinase C inhibitory activity. In T. thermophilus the 321bp gene has a GC content of 67% overall and 94.4% in the third nucleotide position, with unusually no thymine as a wobble base. The gene product, a small highly conserved 11996Da predicted soluble cytoplasmic protein, offers an ideal opportunity to investigate thermostabilising amino acid substitutions. Here we report on the characterisation of the novel hint sequence.