CHLOROPLAST GENE STRUCTURE AND REGULATION
CHLOROPLAST GENE STRUCTURE AND REGULATION
批准号:
3278556
负责人:
NAM-HAI CHUA
金额:
$16.43万
依托单位:
依托单位国家:
美国
项目类别:
财政年份:
1982
资助国家:
美国
项目状态:
已结题
起止时间:
1982-05-01 至 1987-04-30
中文摘要
大多数,如果不是全部的话,叶绿体(ct)中的多聚体酶是由
其结构基因分散在两个不同的细胞中,
隔室(核和CT)。 我们的长期目标是了解
这些基因的表达是如何协调和调节的。 作为
为了实现这一目标,我们希望收集更多关于罚款的数据。
ct基因本身的结构。 尽管ct DNA有可能
编码超过100种多肽,其中只有两种已被确定
迄今 本提案涉及对
编码光系统(PS)多肽亚基的黄瓜ct基因
II复合体。 在ct内产生的多肽亚基
在不存在或存在特异性
抑制剂的 抗体会针对ct合成的亚基
其可能是CT基因产物。 黄瓜的限制性片段
ct DNA将用于从总ct RNA中选择特异性mRNA
混合物. 选择的mRNA将在网织红细胞裂解物中翻译
系统和通过抗体免疫沉淀鉴定的产物
至PS II多肽。 这些实验将确定ct基因编码
PS II多肽,并将其定位于PS II多肽的特异性限制性片段。
已知的地图位置 编码PS II的每个mRNA的5'起始位点
多肽将通过S1技术的修改来作图。 每个
基因的特征在于其转录方向
以及它是否含有内含子。 基因的3'和5'区
将通过Maxam-Gilbert技术进行测序,看看是否有
在基因调控中可能很重要的共有序列。 学习ct
基因表达和调控在体外,我们将建立一个DNA依赖的
使用质体裂解物的可溶性转录系统。 截短的
羧化酶大亚基(LS)基因和32 kd多肽基因将
用作模板。 我们预测这两个基因在ct中的转录
裂解物中,但只有LS基因的原生质体裂解物。 我们希望利用
两种质体裂解物的不同转录反应
隔离类型是一个特定要求的因素,
CT中32 kD多肽基因的转录。
英文摘要
Most, if not all, multimeric enzymes in the chloroplast(ct) are made up of
subunits whose structural genes are dispersed in two different cellular
compartments (nucleus and ct). Our long term objective is to understand
how the expression of these genes are coordinated and regulated. As a
first step toward this goal, we wish to gather more data on the fine
structures of ct genes themselves. Although ct DNA has the potential to
code for more than 100 polypeptides, only two of them have been identified
so far. This proposal deals with the identification and characterization
of cucumber ct genes encoding polypeptide subunits of the photosystem (PS)
II complex. Polypeptide subunits that are made inside the ct will be
determined by pulse-labeling in the absence or presence of specific
inhibitors. Antibodies will be raised against the ct-synthesized subunits
which are likely to be ct gene products. Restriction fragments of cucumber
ct DNA will be used to select for specific mRNA from a total ct RNA
mixture. The selected mRNA will be translated in a reticulocyte lysate
system and the products identified by immunoprecipitation with antibodies
to PS II polypeptides. These experiments will identify ct genes encoding
PS II polypeptides and localize them to specific restriction fragments of
known map positions. The 5' initiation site of each mRNA encoding PS II
polypeptide will be mapped by a modification of the S1 technique. Each
gene will be characterized with respect to its direction of transcription
and whether it contains any introns. The 3' and 5' regions of the gene
will be sequenced by the Maxam-Gilbert technique to see if there are
consensus sequences that may be important in gene regulation. To study ct
gene expression and regulation in vitro we will establish a DNA-dependent
soluble transcription system using plastid lysates. Truncated fragments of
the carboxylase large subunit (LS) gene and the 32 kd polypeptide gene will
be used as templates. We predict transcription of both genes in the ct
lysate but only the LS gene in the etioplast lysate. We hope to exploit
this differential transcriptional response of lysates from the two plastid
types of isolate a factor that is specifically required for the
transcription of the 32 kd polypeptide gene in ct.
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海外基金