Characterization of Three Novel Human Cadherin Genes (CDH7, CDH19, and CDH20) Clustered on Chromosome 18q22–q23 and with High Homology to Chicken Cadherin-7

Full-length coding sequences of two novel human cadherin cDNAs were obtained by sequence analysis of several EST clones and 5′ and 3′ rapid amplification of cDNA ends (RACE) products. Exons for a third cDNA sequence were identified in a public-domain human genomic sequence, and the coding sequence was completed by 3′ RACE. One of the sequences (CDH7L1, HGMW-approved gene symbol CDH7) is so similar to chicken cadherin-7 gene that we consider it to be the human orthologue. In contrast, the published partial sequence of human cadherin-7 is identical to our second cadherin sequence (CDH7L2), for which we propose CDH19 as the new name. The third sequence (CDH7L3, HGMW-approved gene symbol CDH20) is almost identical to the mouse “cadherin-7” cDNA. According to phylogenetic analysis, this mouse cadherin-7 and its here presented human homologue are most likely the orthologues of Xenopus F-cadherin. These novel human genes, CDH7, CDH19, and CDH20, are localized on chromosome 18q22–q23, distal of both the gene CDH2 (18q11) encoding N-cadherin and the locus of the six desmosomal cadherin genes (18q12). Based on genetic linkage maps, this genomic region is close to the region to which Paget's disease was linked. Interestingly, the expression patterns of these three closely related cadherins are strikingly different.     

Characterisation of the beta-tubulin gene of Cylicocyclus nassatus

mRNA and genomic DNA were isolated from adult Cylicocyclus nassatus, and the mRNA was reverse transcribed. The cDNA was PCR amplified using degenerate primers designed according to the alignment of the β-tubulin amino acid sequences of other species. To complete the coding sequence, the 3′ end was amplified with the 3′-RACE, and for amplification of the 5′ end the SL1-primer was used. The cDNA of the β-tubulin gene of C. nassatus spans 1429 bp and encodes a protein of 448 amino acids. Specific primers were developed from the cDNA sequence to amplify the genomic DNA sequence and to analyse the genomic organisation of the β-tubulin gene. The complete sequence of the genomic DNA of the β-tubulin gene of C. nassatus has a size of 2652 bp and is organised into nine exons and eight introns. The identities with the exons of the gru-1 β-tubulin gene of Haemonchus contortus range between 79% and 97%.     

Characterization of thePac25I Restriction-Modification Genes Isolated from the Endogenous pRA2 Plasmid ofPseudomonas alcaligenesNCIB 9867

Genes for the class IIPseudomonas alcaligenesNCIB 9867 restriction-modification (R-M) system,Pac25I, have been cloned from its 33-kb endogenous plasmid, pRA2. ThePac25I endonuclease and methylase genes were found to be aligned in a head-to-tail orientation with the methylase gene preceding and overlapping the endonuclease gene by 1 bp. The deduced amino acid sequence of thePac25I methylase revealed significant similarity with theXcyI,XmaI,Cfr9I, andSmaI methylases. High sequence similarity was displayed between thePac25I endonuclease and theXcyI,XmaI, andCfr9I endonucleases which cleave between the external cytosines of the recognition sequence (i.e., 5′-C↓CCGGG-3′) and are thus perfect isoschizomers. However, no sequence similarity was detected between thePac25I endonuclease and theSmaI endonuclease which cleaves between the internal CpG of the recognition sequence (i.e., 5′-CCC↓GGG-3′). Both thePac25I methylase and endonuclease were expressed inEscherichia coli.An open reading frame encoding a protein which shows significant similarity to invertases and resolvases was located immediately upstream of thePac25I R-M operon. In addition, a transposon designated Tn5563was located 1531 bp downstream of the R-M genes. The location on a self-transmissible plasmid as well as the close association with genes involved in DNA mobility suggests horizontal transfer as a possible mode of distribution of this family of R-M genes in various bacteria.     

Cloning and Gene Mapping of the Mouse Homologue of theCBFA2T1Gene Associated with Human Acute Myeloid Leukemia

The humanCBFA2T1(also known asMTG8) gene, on chromosome 8, has been identified through its involvement in the t(8;21) chromosomal translocation, frequently found in acute myeloid leukemia. We report here the isolation and characterization of the mouse homologue of theCBFA2T1gene,Cbfa2t1h.Nucleotide sequence analysis ofCbfa2t1hcDNA clones revealed an open reading frame encoding a protein of 577 amino acids with an extremely high degree of amino acid identity (99.3%) to the human protein. The nucleotide sequence is also highly conserved between mouse and human in the 5′- and 3′-untranslated regions (87.0, 92.0, and 93.7% identities for 5′-untranslated, coding, 3′-untranslated regions, respectively). The 3′-untranslated region ofCbfa2t1hcontains a (CA)_ndinucleotide repeat, and the polymerase chain reaction amplification of the (CA)_nrepeat region revealed fragment length polymorphism among mouse strains. Using this polymorphism, we have mappedCbfa2t1hto mouse chromosome 4 close to the centromere using SMXA recombinant inbred strains and 106 intersubspecific backcross progenies of the (DBA/2 × Mae) × Mae cross. The chromosomal location was also confirmed by fluorescencein situhybridization.     

Genomic Cloning and Characterization of a Novel Lectin Gene from <Emphasis Type="Italic">Zantedeschia aethiopica</Emphasis>

A new lectin gene was isolated by using genomic walker technology and revealed to encode a mannose-binding lectin. Analysis of a 2233 bp segment revealed a gene including a 1169 bp 5′ flanking region, a 417 bp open reading frame (ORF) and a 649 bp 3′ flanking region. There are two putative TATA boxes and eight possible CAAT boxes lie in the 5′ flanking region. The ORF encodes a 15.1 kDa precursor, which contains a 24-amino acid signal peptide. One possible polyadenylation signal is found in the 3′-flanking region. No intron was detected within the region of genomic sequence corresponding to zaa (Zantedeschia aethiopica agglutinin) full-length cDNA, which is typical of other mannose-binding lectin gene that have been reported. The deduced amino acid sequence of the lectin gene coding region shares 49–54% homology with other known lectins. The cloning of this new lectin gene will allow us to further study its structure, expression and regulation mechanisms.     

Characterization of a Variant ofAutographa californicaNuclear Polyhedrosis Virus with a Nonfunctional ORF 603

A new genotypic variant ofAutographa californicanuclear polyhedrosis virus (AcMNPV), the V8 variant, was originally identified by an additionalHindIII site in theHindIII–F fragment. Insect bioassays of this variant displayed a decreased time of mortality compared with the L1 variant of AcMNPV inSpodoptera frugiperdalarvae but not inTrichoplusia nilarvae. A 1.8-kb region containing the 3′ end of ORF 5,lef-2,ORF 603, and the 5′ end of the polyhedrin gene (polh) of both L1 and V8 was sequenced. V8 exhibited extensive sequence variation in the region between the 3′ end oflef-2and the 5′ end ofpolh; V8 had six amino acid substitutions in thelef-2gene product and a nonfunctional ORF 603. A site-specific frameshift mutation in ORF 603 of the L1 variant was constructed to determine the effect of ORF 603 inS. frugiperdalarvae. Truncation of ORF 603 was found to decrease the time of mortality inS. frugiperdalarvae. The insect-selective toxin gene,tox34, was inserted into the V8 variant by direct cloning. The efficacy of this recombinant as a biopesticide was equivalent to similar L1 recombinants.     

Identification and Characterization of a Seven Transmembrane Hormone Receptor Using Differential Display

Targeted mutagenesis analysis has shown that theCmybproto-oncogene, which encodes a sequence-specific DNA binding protein, is required for normal murine fetal liver erythropoiesis and myelopoiesis. To identify novel genes involved in hematopoiesis, differential display analysis was conducted using total liver RNA isolated from 14.5-day postcoitusCmybwildtype, heterozygous, and homozygous mutant littermates. Using 4 oligo(dT) 3′ primers and 5 arbitrary decamers as 5′ primers, 22 differentially expressed genes have been identified. Eight putatively novel genes were identified from 12 cDNAs that were sequenced. One gene, initially designated DD7A5-7, is primarily expressed in cells of the myeloid lineage. The full-length DD7A5-7 cDNA is 3239 nucleotides, encoding a putative protein of 931 amino acids. The protein is a member of a family of hormone receptors containing 7 transmembrane segments. The receptor also contains 7 epidermal growth factor-like (Egf-like) motifs at the amino terminal of the predicted protein. The gene is alternatively spliced, resulting in the deletion of one or more copies of theEgf-like motif. DD7A5-7 maps to mouse Chromosome 17 and is the putative homologue ofEMR1,a recently describedEgf-like module containing mucin-like hormone receptor with 7 transmembrane segments in humans. Our results indicate that theCmybmutant fetuses represent a unique resource for identifying genes involved in hematopoiesis.     

Molecular cloning and expression analysis of phospholipase Cdelta from mud loach, Misgurnus mizolepis

A gene encoding phosphoinositide-specific phospholipase C (PLC), designated ML-PLCδ, was cloned from mud loach (Misgurnus mizolepis) liver. A complete cDNA encoding ML-PLCδ was isolated by screening the cDNA library of mud loach liver and using the 5′-rapid amplification of cDNA ends (RACE) method. The full-length ML-PLCδ gene contains an open reading frame of 2325 base pairs encoding a 774 amino acid protein with a molecular mass of 88,072 Da; this corresponds to the size of the protein expressed in Escherichia coli BL21 (DE3) using pET28a vector. It contains all of the characteristic domains found in mammalian PLCδ isozymes (PH domain, EF-hands, X–Y catalytic region, and a C2 domain). A homology search revealed that ML-PLCδ shares relatively high sequence identity with mammalian PLCδ1 (51–52%) and catfish PLCδ (64%). The recombinant ML-PLCδ protein expressed as a histidine-tagged fusion protein in E. coli was purified to apparent homogeneity by Ni²⁺-NTA affinity chromatography. The recombinant ML-PLCδ showed a concentration-dependent PLC activity to phosphatidylinositol 4,5-bis-phosphate (PIP₂) and its activity was Ca²⁺-dependent, which was similar to mammalian PLCδ isozymes.