Molecular analysis of the Aedes aegypti carboxypeptidase gene family

To gain a better understanding of coordinate regulation of protease gene expression in the mosquito midgut, we undertook a comprehensive molecular study of digestive carboxypeptidases in Aedes aegypti. Through a combination of cDNA cloning using degenerate PCR primers, and database mining of the recently completed A. aegypti genome, we cloned and characterized 18 A. aegypti carboxypeptidase genes. Bioinformatic analysis revealed that 11 of these genes belong to the carboxypeptidase A family (AaCPA-I through AaCPA-XI), and seven to the carboxypeptidase B gene family (AaCPB-I through AaCPB-VII). Phylogenetic analysis of 32 mosquito carboxypeptidases from five different species indicated that most of the sequence divergence in the carboxypeptidase gene family occurred prior to the separation of Aedes and Anopheles mosquito lineages. Unlike the CPA genes that are scattered throughout the A. aegypti genome, six of seven CPB genes were found to be located within a single 120 kb genome contig, suggesting that they most likely arose from multiple gene duplication events. Quantitative expression analysis revealed that 11 of the A. aegypti carboxypeptidase genes were induced up to 40-fold in the midgut in response to blood meal feeding, with peak expression times ranging from 3 to 36 h post-feeding depending on the gene.     

A novel rat carboxypeptidase, CPA2: characterization, molecular cloning, and evolutionary implications on substrate specificity in the carboxypeptidase gene family

A new member of the carboxypeptidase gene family, carboxypeptidase A2 (CPA2), has been identified from the predicted amino acid sequence of a rat pancreatic cDNA clone. In vivo recombination and in situ hybridization techniques employing the CPA2 cDNA resulted in the isolation of two genomic clones spanning the 25-kilobase pair rat CPA2 gene. Evolutionary trees built from the amino acid sequences of the known pancreatic carboxypeptidases show that CPA2 and carboxypeptidase A1 (CPA1) are the products of genes which duplicated before the mammalian radiation, and that bovine CPA is of the A1 type. The substrate specificities of CPA1 and CPA2 isolated from rat pancreas are similar to bovine CPA in that carboxyl-terminal amino acids with aromatic or branched aliphatic side chains are preferred. However, the substrate preference of rat CPA1 is skewed toward smaller amino acids, while that of rat CPA2 is skewed toward bulkier amino acids as compared to bovine CPA. The differences in the substrate specificities of these three carboxypeptidases are compatible with the nature of the amino acid replacements in their binding pockets for the carboxylterminal amino acid of the substrate.     

Isolation and molecular cloning of mast cell carboxypeptidase A. A novel member of the carboxypeptidase gene family

Mast cell carboxypeptidase A has been isolated from the secretory granules of mouse peritoneal connective tissue mast cells (CTMC) and from a mouse Kirsten sarcoma virus-immortalized mast cell line (KiSV-MC), and a cDNA that encodes this exopeptidase has been cloned from a KiSV-MC-derived cDNA library. KiSV-MC-derived mast cell carboxypeptidase A was purified with a potato-derived carboxypeptidase-inhibitor affinity column and was found by analytical sodium dodecyl sulfate-polyacrylamide gel electrophoresis to be a Mr 36,000 protein. Secretory granule proteins from KiSV-MC and from mouse peritoneal CTMC were then resolved by preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transblotted to polyvinylidine difluoride membranes. Identical aminoterminal amino acid sequences were obtained for the prominent Mr 36,000 protein present in the granules of both cell types. Based on the amino-terminal sequence, an oligonucleotide probe was synthesized and used to isolate a 1,470-base pair cDNA that encodes this mouse exopeptidase. The deduced amino acid sequence revealed that, after cleavage of a 15-amino acid hydrophobic signal peptide and a 94-amino acid activation peptide from a 417-amino acid preproenzyme, the mature mast cell carboxypeptidase A protein core has a predicted Mr of 35,780 and a high positive charge [Lys + Arg) - (Asp + Glu) = 17) at neutral pH. Although critical zinc-binding amino acids (His67, Glu70, His195), substrate-binding amino acids (Arg69, Asn142, Arg143, Tyr197, Asp255, Phe278), and cysteine residues that participate in intrachain disulfide bonds (Cys64-Cys77, Cys136-Cys159) of pancreatic carboxypeptidases were also present in mast cell carboxypeptidase A, the overall amino acid sequence identities for mouse mast cell carboxypeptidase A relative to rat pancreatic carboxypeptidases A1, A2, and B were only 43, 41, and 53%, respectively. RNA and DNA blot analyses revealed that mouse peritoneal CTMC, KiSV-MC, and bone marrow-derived mast cells all express a prominent 1.5-kilobase mast cell carboxypeptidase A mRNA which is transcribed from a single gene. We conclude that mouse mast cell carboxypeptidase A is a prominent secretory granule enzyme of mast cells of the CTMC subclass and represents a novel addition to the carboxypeptidase gene family.     

Structural characterization of exon 6 of the rat IGF-I gene.

In rat liver, insulin-like growth factor I (IGF-I) mRNAs exist as two major size classes of 7.5-7.0 kb and 1.2-0.9 kb. The 7.5- to 7.0-kb IGF-I mRNAs predominate in some nonhepatic tissues of the rat. Because the previously reported sequences of rat IGF-I cDNAs and genomic clones account for only 2.1 kb of sequence, the majority of the sequence of 7.5- to 7.0-kb rat IGF-I mRNAs was unknown. Using a combination of nucleotide sequencing of genomic DNA and cDNA clones and Northern hybridization and RNase protection, we have characterized a 6,354-base-long 3' exon (exon 6) of the rat IGF-I gene. The sequence of exon 6 establishes the previously unknown sequence of the 3' end of the 7.5- to 7.0-kb rat IGF-I mRNAs, comprised predominantly of an unusually long 3' untranslated sequence (3'UT). The long 3'UT contains multiple ATTTA, A(T)nA, and (T)nA sequences, as well as inverted repeats. These sequences may contribute to the shorter half-life of the 7.5- to 7.0-kb rat IGF-I mRNAs relative to the 1.2- to 0.9-kb forms that have been demonstrated previously in vitro and in vivo. We also demonstrate that the 7.5- to 7.0-kb rat IGF-I mRNAs are localized to the cytoplasm of rat liver, providing indirect evidence that they are mature and functional mRNAs.     

Structural analysis of gene loci for rat U1 small nuclear RNA.

Four phage clones which hybridize with U1 small nuclear RNA were obtained from a rat gene library. Two clones contain a presumed pseudogene. A third clone includes two gene candidates that are co-linear with the rat U1-RNA, 3.6kb apart and in the opposite orientation. The two genes are surrounded by identical sequences of 491bp upstream and 178bp downstream. The upstream sequences do not contain a TATA box, but share many block homologies with those for the human U1-RNA gene(1-3). A 101bp "identifier (ID) sequence", which was reported to be specifically expressed in rat brain (4), is inserted immediately after the shared sequence downstream of one of the genes. In the fourth clone, there are two putative pseudogenes, which have one or three nucleotide changes, 3kb apart and in the same orientation. Southern blot analysis of total rat DNA reveals about 50 U1-RNA genes/pseudogenes in the genome.     

Structural and functional analysis of CCT family genes in pigeonpea

Molecular Biology Reports - Pigeonpea (Cajanus cajan L.) is a photoperiod-sensitive short-day plant. Understanding the flowering-related genes is critical to developing photoperiod insensitive...     

Structural and functional analysis of the rat malic enzyme gene promoter.

We have characterized sequences of genomic DNA 5' to the coding region of the rat malic enzyme gene. This sequence possesses neither TATA nor CCAAT sequences in their usual positions but is rich in GC residues. Sequences similar to those found in the regulatory regions of other genes are discussed. Deletion analyses have revealed that sequences +1 to -41 are sufficient to initiate expression, although inclusion of information up to -177 is necessary for maximal promoter activity.     

Evolution of the HLA-DR1 gene family. Structural and functional analysis of the new allele "DR-BON".

The molecules of the MHC are highly polymorphic and involve in Ag presentation; their striking genetic polymorphism allows probable interactions with a large variety of antigenic fragments when these are presented to the TCR. It is therefore of interest to explore the extent of this polymorphism and the mechanisms of its generation. We have studied the class II HLA-DR blank allele DR-BON that has been previously defined by MLR, restriction fragment length polymorphism, and two-dimensional gel electrophoresis. A cDNA library was constructed from a DR-BON homozygous typing cell and cDNA corresponding to DR alpha- and DR beta-chains were sequenced. By comparison with other known alpha- and beta-chain sequences it is shown that the alpha-chain is invariant and the beta-chain differs from DR1 by only six nucleotides, clustered in the third variable region with three amino acid changes at position 67, 70, and 71. The short DNA stretch of sequence encoding the 67-71 region is also present in other DR alleles: DR4/Dw10, DRw13, and some DRw11 specificities. Therefore we propose that a gene conversion-like event has occurred between the DRB1 *0101 (DR1/Dw1) gene and one of these three DRB1 genes. Extensive typing has been performed with a DR-BON-specific 17-mer oligonucleotide. Cross-hybridization with other genes than the ones expected from DNA sequence comparison was not observed. A selected panel of DR-BON reactive T cell clones shows three patterns of reactivity. Some clones are strictly DR-BON specific; some cross-reacted with DRw13 and a few cross-reacted with other haplotypes. The role of different epitopes of the third variable region of HLA-DR beta chain in allo-reaction is discussed.