An Intron Sequence is Present in the 3 UTR of Tomato Cf9 Gene

Tomato leaf mould-resistance gene Cf) was amplified by PCR and cloned from the genomic DNA of a tomato ( Lycopersicon esculentum Mill. ) cultivar ("Zhongza 9'). Sequence analysis indicates that this Cf9 gene is 2 751 bp long and contains an open reading frame encoding a protein of 863 amino acids. An intron of 115 bp in length is found in the 3' untranslated region of this gene. The exon/intron borders are replicate sequences, TCCAGG(T)ATTC, which shares high homology with those of the two genes of Cf2 which is another leaf mould-resistant gene locus in tomato. Compared with the previously reported cDNA sequence of Cf9, a single change of T to C occurs at the nucleotide position of 371 in this PCR-ampliiied Cf) gene, which converts Leu 121 to Pro in the LRR region of the encoded protein.     

Isolation and complete nucleotide sequence of the gene for bovine parathyroid hormone

The structure of the bovine parathyroid hormone (PTH) gene has been analyzed by Southern blot hybridization of genomic DNA and by nucleotide sequence analysis of a cloned PTH gene. In the Southern analysis, several restriction enzymes produced single fragments that hybridized to PTH cDNA suggesting that there is a single bovine PTH gene. The restriction map of the cloned gene is the same as that determined by Southern blot analysis of bovine DNA. The sequence of 3154 bp of the cloned gene has been determined including 510 bp and 139 bp in the 5' and 3' flanking regions, respectively. The gene contains two introns which separate three exons that code primarily for: (i) the 5' untranslated region, (ii) the pre-sequence of preProPTH, and (iii) PTH and the 3' untranslated region. The gene contains 68% A + T and unusually long stretches of 100- to 150-bp sequences containing alternating A and T nucleotides in the 5' flanking region and intron A. The 5' flanking region contains two TATA sequences, both of which appear to be functional as determined by S1 nuclease mapping. Compared to the rat and human genes, the locations of the introns are identical but the sizes differ. Comparable human and bovine sequences in the flanking regions and introns are about 80% homologous.     

The rat casein multigene family. Fine structure and evolution of the beta-casein gene

Eight overlapping phage clones, spanning 34.4 kilobase pairs of genomic DNA, containing the 7.2-kilobase pair rat beta-casein gene have been isolated and characterized. The first 510 base pairs (bp) of 5' flanking, 110 bp of 3' flanking, and all the exon/intron junctions have been sequenced. The beta-casein gene contains 9 exons ranging in size from 21 to 525 bp. We have attempted to identify potential regulatory elements by searching for regions of sequence homology shared between milk protein genes which respond similarly to lactogenic hormones and by searching for previously reported hormone receptor-binding sites. Within the conserved first 200 bp of 5' flanking sequences 3 regions of greater than 70% homology were observed between the rat beta- and gamma-casein genes. One of these contains a region 90% homologous to the chicken progesterone receptor-binding site. The conserved 5' noncoding region, the highly conserved signal peptide, and the hydrophobic carboxyl-terminal region of the protein are each encoded by a separate exon. In contrast the evolutionarily conserved phosphorylation site of beta-casein is formed by an RNA-splicing event. The exons which encode the phosphorylation sites of beta-casein appear to have resulted from an intragenic duplication. Based upon the exon structure of the casein genes, an evolutionary model of intragenic and intergenic exon duplications for this gene family is proposed.     

Structure, chromosome location, and expression of the human smooth muscle (enteric type) gamma-actin gene: evolution of six human actin genes.

Recombinant phages that carry the human smooth muscle (enteric type) gamma-actin gene were isolated from human genomic DNA libraries. The amino acid sequence deduced from the nucleotide sequence matches those of cDNAs but differs from the protein sequence previously reported at one amino acid position, codon 359. The gene containing one 5' untranslated exon and eight coding exons extends for 27 kb on human chromosome 2. The intron between codons 84 and 85 (site 3) is unique to the two smooth muscle actin genes. In the 5' flanking region, there are several CArG boxes and E boxes, which are regulatory elements in some muscle-specific genes. Hybridization with the 3' untranslated region, which is specific for the human smooth muscle gamma-actin gene, suggests the single gene in the human genome and specific expressions in enteric and aortic tissues. From characterized molecular structures of the six human actin isoform genes, we propose a hypothesis of evolutionary pathway of the actin gene family. A presumed ancestral actin gene had introns at least sites 1, 2, and 4 through 8. Cytoplasmic actin genes may have directly evolved from it through loss of introns at sites 5 and 6. However, through duplication of the ancestral actin gene with substitutions of many amino acids, a prototype of muscle actin genes had been created. Subsequently, striated muscle actin and smooth muscle actin genes may have evolved from this prototype by loss of an intron at site 4 and acquisition of a new intron at site 3, respectively.     

The structure of the rat ameloblastin gene and its expression in amelogenesis

Ameloblastin (also designated amelin and sheathlin) is an enamel matrix protein expressed within the ameloblast lineage. In this study we analyzed the structure of the rat ameloblastin gene and characterized its subtypes. The promoter sequence contains several E-box-like elements, and consensus sequences for AP1 and SP1. The gene is about 6 kb in length and contains 12 exons. Exon 1 was mapped by primer extension and encodes 90 bp of 5' untranslated leader sequence, followed by the coding sequences of exon 2 (309 bp), alternatively spliced exon 3a (45 bp), exon 3b (198 bp), exon 4 (36 bp), exon 5 (60 bp), exon 6 (45 bp), exon 7 (150 bp), and exon 8 (448 bp) containing coding sequence (426 bp) and 3' untranslated sequence (22 bp), followed by exon 9 (39 bp); exon 10 (143 bp); exon 11 (342 bp); and exon 12. Exon 3a, encoding YEYSLPVHPPPLPSQ, has a potential SH3 binding domain. Analysis of ameloblastin subclones showed that exon 3a and 11 were potential alternative splicing sites, producing 4 types of ameloblastin mRNA, from which ameloblastin I and II could be translated. Using in situ hybridization, immunohistochemistry, Western blot and RT-PCR methods we found that ameloblastin II, containing exon 3a, was more strongly expressed at the late maturation stage of ameloblasts than at the secretory stage, while a common probe for both ameloblastin subtypes showed wide expression throughout the presecretory, secretory and postsecretory stages. From the above results we propose that ameloblastin II plays an important role in the mineralization of ameloblasts during the maturation stages.     

Structure of the murine mb-1 gene encoding a putative sIgM-associated molecule

Genomic DNA clones containing the B cell-specific murine mb-1 gene were isolated and a 5.6-kb BamH I fragment was characterized. It is 5629 bp long and contains five exons: an exon containing the 5' untranslated and the coding sequence of the signal peptide, an exon of 294 bp, which contains most of the extracellular sequence of the MB-1 protein, a 119-bp long exon coding mainly for the transmembrane portion, and two exons of 69 bp and 427 bp encoding the cytoplasmic domain and the 3'-untranslated region, respectively. The mb-1 gene does not contain a "TATA box" found in many eukaryotic promoters. The 5'-flanking region has sequence stretches homologous to IgVH 5'-promoter regions and a bcl 2 intron sequence. It contains the decanucleotide sequence (ATGGCAAATA) almost identical to the octamer motif of IgVH promoters. A B cell-specific DNase I-hypersensitive site was found in the 3'-flanking region indicating that this region might be involved in B cell-specific expression of mb-1. Southern blot analysis of genomic liver DNA with the cloned mb-1 cDNA suggests the existence of another mb-1-related gene segment.     

Isolation and characterisation of genomic and cDNA clones for an androgen-regulated secretory protein of rat seminal vesicles.

Testosterone controls the synthesis of seminal vesicle protein F in male rats by regulating the cellular concentration of its mRNA (mRNAF). Phage lambda recombinants have been isolated containing the complete F gene. In addition plasmids have been constructed containing cDNAF sequences some of which are probably full-length (approximately 700 bp). Detailed restriction mapping shows that the F gene is 1.7 kbp long and contains approximately 1.0 kbp of intervening sequence arranged in at least two introns (420 bp and 600 bp). Part of cDNAF has been sequenced showing that the terminal 125 bp of the 3' untranslated region of mRNAF has substantial (greater than 70%) sequence homology with the 3' end of the mRNA coding for another androgen-dependent seminal vesicle protein (protein S). The cloned F gene has been detected in liver and seminal vesicle DNA along with an homologous but structurally different gene. The hormonal control of mRNAF was examined with cDNAF. A pronounced (approximately 3000-fold) differential response to testosterone was observed.