小米psbA基因5''末端非编码区的结构特征

以小米(Setaria italica)为材料,克隆了含有叶绿体psbA基因的2.2kb EcoRⅠ片段,测定了该基因5＇末端非编码区的核苷酸序列。序列分析显示psbA基因5＇末端非编码区存在着与原核类似的启动子结构,其“-10”区的序列为TATACT,与原核生物“-10”共有基序(Consensus motif)仅相差一个核苷酸;其“-35”区的序列为TTGACA,与原核生物“-35”共有基序完全相同。另外,在“-10”区和“-35”区之间还存在着一个类似真核启动子结构的“TATATA”保守序列。这些结果表明小米psbA基因的启动子既具有原核的特征又具有真核的特征。小米psbA基因的mRNA前导序列长87bp,与高粱完全一致,而比水稻多出了“CTATTTT”7个核苷酸,比小麦、大麦和黑麦多出了“TTTT”4个核苷酸。因此推测在禾本科的C_3和C_4植物之间,psbA基因mRNA前导序列区的差异可能具有普遍性。计算机分析结果显示,以上6种植物的psbA基因mRNA前导序列区内均能形成小的茎环结构,而且这段“CTATTTT”额外序列恰好位于茎环结构中,造成了6种植物间茎环大小的差异。这一小的二级结构可能对psbA基因的表达调控有一定的影响。     

Structural feature of sorghum chloroplastpsbA gene and regulation effects of its 5′-noncoding region

Comparative analysis reveals remarkahle homology between the sequences of bothpsbA gene nucleotides and the inferred amino acids of sorghum, a C₄ plant, and those of rice, a C₃ plant. The 5′-noncoding region of sorghumpsbA gene contains the conservative promoter elements, “—35” element and “—10” element, like the prokaryote and the promoter element, TATA box, like the eukaryote. As compared with that of the rice, an extra sequence of 7 bp is found in the leader sequence of the mRNA in the former. Using anin vitro system, it has been demonstrated that protein factor exists in sorghum chloroplast protein extract which specifically hinds to the 5′-noncoding region ofpsbA gene. Measurement of the expression of luciferase shows a 2–5 time greater reaction of the expression plasmids pALqs which contain leader region of sorghumpsbA gene than that of the expression plasmids pALqr which contain leader region of ricepsbA gene inE. coli.     

Structural feature of sorghum chloroplast psbA gene and regulation effects of its 5'-noncoding region*

Comparative analysis reveals remarkahle homology between the sequences of bothpsbA gene nucleotides and the inferred amino acids of sorghum, a C₄ plant, and those of rice, a C₃ plant. The 5′-noncoding region of sorghumpsbA gene contains the conservative promoter elements, “—35” element and “—10” element, like the prokaryote and the promoter element, TATA box, like the eukaryote. As compared with that of the rice, an extra sequence of 7 bp is found in the leader sequence of the mRNA in the former. Using anin vitro system, it has been demonstrated that protein factor exists in sorghum chloroplast protein extract which specifically hinds to the 5′-noncoding region ofpsbA gene. Measurement of the expression of luciferase shows a 2–5 time greater reaction of the expression plasmids pALqs which contain leader region of sorghumpsbA gene than that of the expression plasmids pALqr which contain leader region of ricepsbA gene inE. coli. Project supported by the Chinese National “863” and “973” Projects     

Analysis of the gB promoter of herpes simplex virus type 1: high-level expression requires both an 89-base-pair promoter fragment and a nontranslated leader sequence.

To investigate the cis-acting sequences involved in regulation of a herpes simplex virus gamma 1 gene, deletion analyses of the glycoprotein B (gB) gene promoter were performed. In transfection assays with gB-chloramphenicol acetyltransferase plasmids, high-level constitutive expression from the gB promoter was found with an 89-bp sequence (-69 to +20). Additional sequences in the 5'-transcribed noncoding leader region (+20 to +136) were required for full stimulation by herpes simplex virus infection. Plasmids with progressive deletions of the gB leader sequence demonstrated that chloramphenicol acetyltransferase expression in infected cells was proportional to the length of the leader region retained. In recombinant viruses containing a gB-gC gene fusion, a similar 83-bp (-60 to +23) region of the gB gene was found to promote accurately initiated gC mRNA from the viral genome with the same kinetics as the wild-type gB gene. Although the kinetics of expression remained the same, RNA abundance was greater with a 298-bp (-260 to +38) promoter than with the 83-bp promoter.     

The Saccharomyces cerevisiae ADE1 gene: structure, overexpression and possible regulation by general amino acid control.

The ADE1 gene of the yeast Saccharomyces cerevisiae has been cloned by complementation of the ade1 mutation. The nucleotide sequence has been determined for the 918-bp coding region, 240-bp 5'-noncoding region and 292-bp 3'-noncoding region. The sequenced region includes a single large open reading frame coding for a protein of 306 amino acid (aa) residues. The promoter of the ADE1 gene contains a copy of the 5'-TGACTC hexanucleotide, a feature characteristic of promoters under general aa control. Subsequent search of other published purine biosynthesis gene sequences revealed that all of them also contain general aa control signals in their promoter regions. An expression plasmid containing the ADE1 coding region under control of the PHO5 promoter produced N-succinyl-5-aminoimidazole-4-carboxamide ribotide (SAICAR) synthetase in yeast cells at a level of 40% of total cellular protein. One-step purification resulted in an almost homogeneous preparation of SAICAR synthetase.     

Promoter elements required for developmental expression of the maize Adh1 gene in transgenic rice.

To define the regions of the maize alcohol dehydrogenase 1 (Adh1) promoter that confer tissue-specific expression, a series of 5' promoter deletions and substitution mutations were linked to the Escherichia coli beta-glucuronidase A (uidA) reporter gene and introduced into rice plants. A region between -140 and -99 not only conferred anaerobically inducible expression in the roots of transgenic plants but was also required for expression in the root cap, embryo, and in endosperm under aerobic conditions. GC-rich (GC-1, GC-2, and GC-3) or GT-rich (GT-1 and GT-2) sequence motifs in this region were necessary for expression in these tissues, as they were in anaerobic expression. Expression in the root cap under aerobic conditions required all the GC- and GT-rich motifs. The GT-1, GC-1, GC-2, and GC-3 motifs, and to a lesser extent the GT-2 motif, were also required for anaerobic responsiveness in rice roots. All elements except the GC-3 motif were needed for endosperm-specific expression. The GC-2 motif and perhaps the GT-1 motif appeared to be the only elements required for high-level expression in the embryos of rice seeds. Promoter regions important for shoot-, embryo-, and pollen-specific expression were proximal to -99, and nucleotides required for shoot-specific expression occurred between positions -72 and -43. Pollen-specific expression required a sequence element outside the promoter region, between +54 and +106 of the untranslated leader, as well as a silencer element in the promoter between -72 and -43.