The structural gene for ferredoxin I, petF, from the cyanobacterium Anabaena sp. strain PCC 7120 has been isolated from a recombinant lambda library. Mixtures of tetradecanucleotides and heptadecanucleotides, each containing all possible DNA sequences corresponding to two separate regions of the ferredoxin amino acid sequence, were synthesized and used as hybridization probes to identify a genomic clone containing the coding sequence for the petF gene. The sequence of the entire petF coding region and portions of the 3'-and 5'-flanking regions was determined. The DNA sequence of petF suggests that, in contrast to the nucleus-encoded plant protein, cyanobacterial apoferredoxin is not synthesized as a higher-molecular-weight precursor. The Anabaena petF gene is a single-copy gene. During growth on complete medium it was transcribed into a monocistronic mRNA species of approximately 500 bases that initiated 100 base pairs upstream from the petF coding region.Ferredoxin is an acidic, nonheme, iron-sulfur protein with a wide distribution in organisms ranging from nonphotosynthetic, anaerobic bacteria to higher plants and animals. In plants and cyanobacteria, ferredoxin is recognized primarily as a component of the photosynthetic electron transport chain (31). In addition to its central role in NADP+ reduction and cyclic photophosphorylation, ferredoxin functions, directly or indirectly, in a variety of biological electron transfer systems. These include such diverse processes as nitrogen fixation (38), sulfate-sulfite reduction (32), nitrate-nitrite reduction (17, 25), and glutamate synthesis (17). Furthermore, ferredoxin is responsible, via the ferredoxinthioredoxin system, for the regulation of enzymes involved in photosynthetic carbon metabolism (3).Two distinct molecular forms of ferredoxin have been identified in some plants and cyanobacteria. The primary structure of ferredoxins I and II from a number of organisms has been determined (for a compilation, see references 12 and 21), and the amino acid sequence data suggest that these two ferredoxins arose via gene duplication (21). At present, however, no functional differences have been attributed to this dimorphism. Aside from ferredoxins I and II, some cyanobacteria apparently contain yet another form of this protein. Cohn et al. (6) have isolated a membrane-associated ferredoxin from the cyanobacterium Aphanizomenon flosaquae that is distinct from ferredoxin I with regard to location within the cell, chromatographic behavior on a DEAE-cellulose column, and electron spin resonance spectra. The limited amino acid sequence data suggest that the membrane-associated ferredoxin is more similar to ferredoxin I than most ferredoxin II molecules are to ferredoxin

A cluster of genes encoding subunits of ATP synthase of Anabaena sp. strain PCC 7120 was cloned, and the nucleotide sequences of the genes were determined. This cluster, denoted atpi, consists of four Fo genes and three F1 genes encoding the subunits a (atpl), c (atpH), b' (atpG), b (atpF), 8 (atpD), a (aptA), and y (atpC) in that order. Closely linked upstream of the ATP synthase subunit genes is an open reading frame denoted gene 1, which is equivalent to the uncI gene of Escherichia coli. The atp1 gene cluster is at least 10 kilobase pairs distant in the genome from apt2, a cluster of genes encoding the (atpB) and E (atpE) subunits of the ATP synthase. This two-clustered ATP synthase gene arrangement is intermediate between those found in chloroplasts and E. coli. A unique feature of the Anabaena atpl cluster is overlap between the coding regions for atpF and atpD. The atpl cluster is transcribed as a single 7-kilobase polycistronic mRNA that initiates 140 base pairs upstream of gene 1. The deduced translation products for the Anabaena sp. strain PCC 7120 subunit genes are more similar to chloroplast ATP synthase subunits than to those of E. coli.The proton-translocating ATP synthase is a multimeric membrane protein complex that couples a transmembrane gradient of electrochemical potential energy produced during electron transport to formation of ATP. This ubiquitous enzyme is found in cell membranes of bacteria, in inner mitochondrial membranes, and in thylakoid membranes of plant chloroplasts (reviewed in references 15, 18, and 30). In all examples studied the enzyme consists of two multimeric components: an extrinsic portion, F1, composed of subunits denoted a, P, -y, 8, and , and in integral membrane portion Fo, composed of several subunits which vary depending on the source of the ATP synthase. In Escherichia coli and chloroplasts, the two systems used for comparison in this study, there are three (a to c) and four (I to IV) Fo subunits, respectively. In E. coli, genes encoding all eight subunits of the ATP synthase are tightly linked and cotranscribed (15). For the ATP synthase of chloroplasts, genes for some subunits are encoded in the nucleus and others are encoded in the organelle genome. The chloroplast ATP synthase genes of higher plants are organized into two separate transcriptional units: the and E genes are linked and cotranscribed (35), while a second cluster containing the I, III, IV, and a. subunit genes map many kilobase pairs away in a second ATP synthase gene cluster (9, 19). The -y, 8, and subunit II genes are nuclear (34).The cyanobacteria are procaryotes with an oxygenevolving photosynthetic system nearly identical to that of plant chloroplasts. The similarity between cyanobacterial and plant photosystems, as well as the procaryotelike features of chloroplasts, lends support to the proposal (28) that * Corresponding author. t Paper no.

Our studies of mutational mechanisms in mammalian cells use the AS52 Chinese hamster ovary cell line. AS52 mutants can be selected as 6-thioguanine resistant colonies and mutations are studied at a chromosomally integrated gpt locus. Mutant gpt sequences are amplified using the polymerase chain reaction (PCR) to distinguish deletions from putative point mutations. PCR is efficiently performed from a few thousand lysed cells or from isolated genomic DNA. Amplified mutant PCR fragments carrying putative point mutations are further characterized by localizing the site of the mutation using chemical modification. A heteroduplex molecule consisting of one wild-type and one mutant DNA strand is generated. A base mismatch will be produced at the site of the mutation. Mismatched cytosine or thymine residues are sensitive to modification by hydroxylamine or osmium tetroxide, respectively. The modified DNA heteroduplex is then sensitive to piperidine cleavage. If one strand is 32P-end labeled, then the cleavage product can be separated on a denaturing acrylamide sequencing gel and visualized using autoradiography. Thus, the site of a mutation can be localized to a specific region of the gene, thereby simplifying the DNA sequence analysis and facilitating the rapid generation of mutational sequence spectra.

No abstract

The duck hepatitis B virus (DHBV) genome contains a pol gene that codes for the viral polymerase protein. This enzyme, which is essential for the replication of the virus, has multiple activities including an RNA directed DNA polymerase or reverse transcriptase (RT) activity, an RNase H activity, and a DNA-directed DNA polymerase activity. The assay described in this chapter is designed to measure the ability of test compounds to inhibit the RNA-directed DNA polymerase activity of the DHBV pol gene product. The assay is based on research performed in the laboratory of Dr. Christoph Seeger (1) and on the observation that the e stem loop structure in the pregenomic RNA is required for initiation of DNA synthesis (2-4).