Isolation and sequencing of mouse angiogenin DNA

The mouse genomic DNA for angiogenin, a potent blood vessel inducing protein, has been isolated from a bacteriophage library using the human angiogenin gene as a probe. The 1129 bp fragment contains 499 bp in the 5' flanking region, 192 bp in the 3' flanking region, and 438 bp coding for the mature protein (121 amino acids) and signal peptide (24 amino acids). Potential TATA box and AATAAA polyadenylation sequences are present, and a consensus sequence for an intron 3' boundary occurs 16 bp upstream of the Met-(24) codon, suggesting the presence of an intron in the 5' region. The protein sequence inferred from the DNA is 76% identical to that of human angiogenin, and matches the sequences obtained previously from tryptic peptides of a serum-derived mouse angiogenin. The critical catalytic residues of human angiogenin are conserved in the mouse protein, as are the six cysteines necessary for disulfide bond formation.     

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.     

Structure and expression of a rice hsp70 gene

A genomic hsp70 gene was isolated from a rice IR36 genomic library and 4 794 bp of the gene have been sequenoed. The 5' flanking region of the gene contained a putative TATA box and a typical heat shock element sequence 5'-CTcgGAAccTTCgAG-3'. The amino acid sequence of the rice HSP70 deduced from the coding region shared 84%-92% homologies with those of HSP70s from other plant species. An intron 1939bp long was identified in the coding region at the codon specifying amino acid 72 (Asp), the similar position introns occurring in other intron-containing hsp70 genes. In addition, another intron of 57 bp was found in the 3'-untranslated region in the rice hsp70 gene. Southern blot hybridization showed that rice hsp70 gene family contained at least three members. Analysis of the RNA leveis with the gene-specific and non-specific probes revealed that the rice hsp70 gene expressed at normal temperature and the expression was enhanced by heat shock treatment.     

Characterization of the rat carbonic anhydrase II gene structure: sequence analysis of the 5′ flanking region and 3′ UTR

The rat carbonic anhydrase II gene was characterized and found to be approximately 15.5 kb in length and to contain 7 exons and 6 introns. All intron/exon junction and branch point sequences conform to consensus sequences, and the overall rat CA II genomic structure appears to be conserved upon comparison with mouse, human, and chicken CA II genes. The putative cis-acting elements within the analyzed 1014 bp 5′ flanking region include: TATA box, 4 Sp1 binding sites, 2 AP2 sites and putative tissue-specific β-globin-like repeat elements. A CpG island of approximately 800 bp was identified that begins about 600 bp upstream of exon 1 and extends about 200 bp into intron 1. In the 3′ UTR, two polyadenylation signals (AATAAA) are present, the second of which is believed to be utilized. Northern blot analysis reveals that the 1.7 kb rat CA II mRNA is abundantly expressed in adult male brain and kidney, while negligible amounts are detected in heart and liver.     

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.     

Cloning and promoter analysis of the cotton lipid transfer protein gene Ltp3

A cotton Ltp3 gene and its 5′ and 3′ flanking regions have been cloned with a PCR-based genomic DNA walking method. The amplified 2.6 kb DNA fragment contains sequences corresponding to GH3 cDNA which has been shown to encode a lipid transfer protein (LTP3). The gene has an intron of 80 bp which is located in the region corresponding to the C-terminus of LTP3. The Ltp3 promoter was systematically analyzed in transgenic tobacco plants by employing the Escherichia coli β-glucuronidase gene (GUS) as a reporter. The results of histochemical and fluorogenic GUS assays indicate that the 5′ flanking region of the Ltp3 gene contains cis-elements conferring the trichome specific activity of Ltp3 promoter.     

家蝇卵黄蛋白基因的克隆与结构序列分析

家蝇卵黄蛋白基因编码的卵黄蛋白是家蝇胚胎发育的重要营养来源 .根据 3种家蝇卵黄蛋白cDNA保守序列设计引物 ,用PCR技术从家蝇基因组DNA中扩增到大小为 76 8bp的mdYP1基因的部分DNA片段 .经地高辛标记成特异性探针 ,从构建的家蝇基因组文库中筛选出一个阳性克隆 ,并从该克隆中分离到大小为 3991bp的mdYP1基因组基因 .序列分析显示 ,该基因组序列含有约1 6kb的 5′ 上游区和 1 0kb的 3′ 下游区 ,编码区由一个 6 1bp的内含子和大小分别为 2 2 2bp和10 2 8bp的 2个外显子组成 .5′ 上游区含有典型的CAAT TATA盒 .     

Genetics and expression of two pectinesterase genes in Valencia orange

The genetics and expression of pectinesterase (PE) genes were examined in Valencia orange. Degenerate primers based on partial amino acid sequence of a 36 kDa PE protein isolated from juice vesicles were used to amplify a 350 bp DNA fragment from cDNA prepared from juice vesicle total RNA. Two groups of 350 bp PE clones with 66% sequence identity were isolated. A clone from each group was used to screen a Valencia orange genomic DNA λ library. Two different lambda clones that contained complete PE coding sequence (CsPME1 and CsPME3) and a third lambda clone that contained partial PE sequence (CsPME2) were characterized. The CsPME1 gene contained two exons (1063 and 689 bp) interrupted by a 1452 bp intron, whereas the CsPME3 gene had two exons (844 and 686 bp) interrupted by a 771 bp intron. CsPME1 shared significant sequence similarity with the partial clone CsPME2, including the entire cloned region of the first exon, a large region in the 5′ portion of the intron and the 3′ portion of the second exon, but the 3′ portion of the intron and the 5′ portion of the second exon were dissimilar. Southern blots suggested that Valencia orange has two genes within each PE group. Full-length cDNA clones that shared 99% sequence identity with CsPME1 and CsPME3 were isolated. Both groups of PE genes were differentially expressed in tissues of Valencia orange, and in addition CsPME3 appeared to be ethylene-regulated. The deduced proteins of PE cDNA clones CsPME1 (63.5 kDa) and CsPME3 (56.3 kDa) were considerably larger than the PE protein we isolated from Valencia orange juice vesicles and also other mature plant PE proteins. The estimated size of group I (2.2 kb) and group II (2.0 kb) PE mRNAs also predicted a larger protein than was isolated from juice vesicles. Alignment of the mature tomato and mung bean PE proteins, the most N-terminal sequence we obtained from polypeptides derived from the 36 kDa PE isolated from juice vesicles and the deduced amino acid sequences of plant PE cDNA clones suggest that a post-translational cleavage event separates the variable N-terminus from the more conserved C-terminal domain of the mature PE protein.     

Isolation and sequence analysis of the genomic DNA fragment encoding an aspartic proteinase inhibitor homologue from potato (Solanum tuberosum L.)

A genomic DNA clone encoding an aspartic proteinase inhibitor of potato was isolated from a lambda EMBL3 phage library using the aspartic proteinase inhibitor cDNA as a hybridization probe. The gene has all characteristic sequences normally found in eucaryotic genes. Typical CAAT and TATA box sequences were found in the 5-upstream region. In this part are also two putative regulatory AGGA box sequences located. In the genomic sequence there are no intron sequences interrupting the coding region. An open reading frame of the gene encodes a precursor protein of 217 amino acids which shows high percent identity with the aspartic proteinase inhibitor cDNA.     

A virus-inducible tobacco gene encoding a glycine-rich protein shares putative regulatory elements with the ribulose bisphosphate carboxylase small subunit gene

cDNA to an mRNA that is strongly induced in Samsun NN tobacco after tobacco mosaic virus (TMV) infection or salicylic acid treatment was used to probe a genomic blot and to screen a genomic library. The mRNA corresponds to a family of approximately eight genes, four of which were cloned. The sequence of the genes and flanking DNA in two clones was determined. One gene was found to contain an intron of 555 bp; S1-nuclease mapping studies indicated that this gene is expressed. The other gene is interrupted by an intron of 1,954 bp and is probably not expressed after TMV infection. The genes encode a protein of 109 amino acids with a putative N-terminal signal peptide of 26 amino acids. The protein contains a high proportion of glycine (25%) and charged amino acids (29%), suggesting that it may be a cell wall component. A comparison of the upstream sequences of the genes encoding the glycine-rich protein and the pathogenesis-related protein 1a showed only limited homology, although both genes are TMV- and salicylic acid-inducible. However, the upstream sequence of the glycine-rich protein gene contains a 64-bp inverted repeat that occurs in a similar position in the tobacco ribulose bisphosphate carboxylase small subunit gene.