DNA sequence organization and transcription of the chicken genome.

A new approach has been used to examine DNA sequence organization in the chicken genome. The interspersion pattern was determined by studying the fraction of labelled DNA fragments of different lengths that hybridized to an excess of short chicken repeated DNA sequences. The results indicate that chicken DNA has a pattern of sequence organization quite different than the standard 'Xenopus' or 'Drosophila' patterns. Two classes of unique sequences are found. One, 34% of the genome, consists of unique sequences approx. 4 kb long interspersed with repeated sequences. The second, non-interspersed fraction, 38% of the genome, consists of unique sequences found in long tracts, a minimum of approx. 22 kb in length. In an attempt to determine whether a relationship exists between DNA sequence organization and the distribution of structural genes we have isolated chicken DNA sequences belonging to different interspersion classes and tested each for the presence of structural genes by hybridization to excess poly(A)+ mRNA. Sequences complementary to poly(A)+ mRNA can be found with approximately the same frequency in both the non-interspersed fraction of the genome and a repeat-contiguous fraction enriched for interspersed sequences.

Repeated sequences in L-cell mRNA complementary to long deoxypolypyrimidines.

Long pyrimidine tracts form part of the repeated DNA sequences in eukaryotic genomes and are among the most highly conserved sequences in evolution. At least some of these sequences are transcribed in L-cells, since they are capable of hybridizing extensively with total cellular RNA. In saturation hybridization experiments involving fractionated cellular RNA, polypyrimidines react preferentially with the polysomal poly(A+) RNA and the nuclear poly (A+) RNA fractions; there is little or no reaction with poly (A-) RNA or with synthetic poly (A). In addition, hybridization between mRNAs and deoxypolypyrimidines is not affected by enzymatic removal of the poly (A+) tract from mRNA molecules. Saturation hybridization experiments indicate that about 1% of mRNA consists of regions complementary to deoxypolypyrimidines. The size of the polypurine regions in mRNA has been measured by gel electrophoresis of RNAase-resistant hybrids and was found to be heterogeneous with an average size of about 35 nucleotides. It is estimated that about half of poly (A-) mRNA molecules in L-cells contain sequences complementary to deoxypolypyrimidines. Although the function of polypyrimidine tracts is not yet known, the conservation and transcription of these sequences suggests an important role in the expression of eukaryotic genes.

Polypyrimidine sequences found in eukaryotic DNA have been conserved during evolution.

L-cell DNA contains an unexpectedly large amount of long pyrimidine tracts. Hydroxyapatite chromatography has been employed to show that these polypyrimidines hydridize extensively to the reiterated DNA of a large number of eukaryotes but fail to hybridize to prokaryotic DNA. This reaction is sequence specific and not the result of a special property of polypyrimidines since random 3H-labelled poly(dC-dT) shows poor hybridization to eukaryotic DNA. The hybrids formed by L-cell polypyrimidines and heterologous repeated sequences have a higher thermal stability than the corresponding hybrids of total repeated DNA indicating that sequences related to these polypyrimidines have been conserved during evolution. Furthermore, at least some of these tracts are transcribed because they are capable of reacting extensively with total cellular RNA. Although the function of these sequences is not yet known, the fact that they are widely conserved in evolution and also transcribed leads us to speculate that they play an an important role in eukaryotic cells.

Organization and transcriptional activity of brain chromatin subunits.

Digestion of brain nuclei with micrococcal nuclease produces 11.2-S chromatin subunits which comprise up to 80% of the total nuclear DNA. Hybridization studies with excess subunit DNA demonstrate that subunits are distributed throughout the repeated and nonrepeated sequences of the genome. No class of nonrepeated DNA appears to be excluded from subunits. Transcribed DNA is present in chromatin subunits since in vitro labelled poly(A+)-mRNA hybridizes to excess subunit DNA with kinetics identical to that for total DNA.

A class-I intron in a cyanelle tRNA gene from Cyanophora paradoxa: phylogenetic relationship between cyanelles and plant chloroplasts.

Cyanelles are photosynthetic organelles which are considered as intermediates between cyanobacteria and chloroplasts, and which have been found in unicellular eukaryotes such as Cyanophora paradoxa. The nucleotide sequence of a 667-bp region of the cyanelle genome from Cyanophora paradoxa containing genes coding for tRNA(UUCGlu) and tRNA(UAALeu) has been determined. The gene coding for tRNA(UAALeu) is split by a 232-bp intron which has a secondary structure typical for class-I structured introns and which is closely related to the intron located in the corresponding gene from liverwort and higher plant chloroplasts. It appears therefore that these tRNA(UAALeu) genes are all derived from one common ancestral gene which already contained a class-I intron.

In vitro production of large single-stranded templates for DNA sequencing.

A method has been developed for the preparation of large single-stranded DNA sequencing templates from primary cloning plasmids or cosmids. The method utilizes the separate action of T7 Gene 6 exonuclease and exonuclease III to generate large quantities of single-stranded template for each strand of a large-cloned fragment. Since the procedure is intended for use on primary clones, it avoids the time-consuming subcloning steps associated with most sequencing programs. The procedure also has the advantage of avoiding clone instability problems associated with subcloning in M13.

Comparative DNA renaturation kinetics in amphibians.

Amphibian haploid genome sizes vary from 9 x 10(8) to 8 x 10(10) nucleotide pairs. The rate of reassociation of DNA from amphibians of different genome sizes has been employed to eliminate one of the theoretical models of chromosome structure. Scaphiopus couchi, Bufo marinus, and Rana clamitans, whose haploid genome sizes are in the ratio 2:7:10, all contain sequences of DNA represented once in the haploid genome. This indicates that their chromosomes are not composed of identical lateral strands (polynemy). The relative frequencies of repetition of DNA sequences are different for the various species of amphibians. The observed frequencies of repetitive DNA sequences in amphibians do not show the relationships expected if amphibians form a polyneme series.

Chlorobenzoate catabolism and interactions between Alcaligenes and Pseudomonas species from Bloody Run Creek.

A mixed community of bacteria from surface runoff waters of the Hyde Park industrial landfill was enriched on 3-chlorobenzoate. Alcaligenes and Pseudomonas species were dominant in the community. Alcaligenes sp. BR60 carried an unstable plasmid specifying 3-chlorobenzoate catabolism. Metabolites detected in culture supernatants included chlorocatechol and chloro-cis, cismuconic acid. Oxygen uptake in the presence of 3- and 4-substituted methyl-catechols revealed a catechol-1,2-oxygenase activity specific for substituted catechols with very limited activity for catechol. The isolate grew very slowly on benzoate. Alcaligenes sp. BR60 was isolated in co-culture with Pseudomonas fluorescens NR52. The latter contained no detectable plasmids and did not grow on benzoate or any of the chlorobenzoates in pure culture. Growth of the co-culture in Bloody Run Creek water supplemented with 3-chlorobenzoate indicated that phosphate concentrations in the water severely limited biodegradation. Under phosphate limited conditions in continuous culture, Pseudomonas fluorescens NR52 effectively scavenged available phosphate when it was present at a ratio of 1 cell to 20 of Alcaligenes sp. BR60. Under these conditions the growth of Alcaligenes sp. BR60 on 3-chlorobenzoate was reduced 5 fold, the frequency of plasmid deletion mutants increased, and 96% of the contaminant remained in the outflow in the form of the starting material or metabolites. No evidence was found for conjugation of the plasmid determining chlorobenzoate catabolism in Alcaligenes sp. BR60 to P. fluorescens NR52.

Characterization of a cyanobacterial iron stress-induced gene similar to psbC.

Recently we have reported that the flavodoxin gene from the cyanobacterium Anacystis nidulans R2 is transcribed as part of an iron stress-induced operon containing multiple mRNA species (D. E. Laudenbach, M. E. Reith, and N. A. Straus, J. Bacteriol. 170: 258-265, 1988). Here we report that nucleotide sequence analyses of DNA located immediately upstream of the flavodoxin gene revealed an open reading frame of 1,026 bases (designated isiA; iron stress inducible) with a deduced amino acid sequence showing similarity to that of the psbC polypeptide of higher plants and cyanobacteria. Assuming proteolytic cleavage of the initial methionine residue, the open reading frame encodes a 341-amino-acid polypeptide with a molecular mass of 36,824 daltons. Amino acid sequence comparisons with known psbC polypeptides from spinach and A. nidulans R2 showed extensive similarity, especially in the proposed membrane-spanning regions. Mung bean nuclease mapping and primer extension experiments have localized a transcriptional start site to a position 19 bases upstream from the first methionine codon of the isiA gene product. The upstream region contains an Escherichia coli-like -10 sequence but lacks the typical -35 consensus sequence. Approximately 15, 25, and 150 bases upstream from the isiA transcription start site are 17 base sequences which resemble the operator sequences of iron-regulated genes of E. coli.

Nucleotide sequence of the chloroplast gene responsible for triazine resistance in canola.

The nucleotide sequence for the psbA gene from a triazine resistant cultivar of B. napus (cv 'Triton') has been determined. This gene encodes an open reading frame of 353 amino acids that is highly homologous to other higher plant psbA genes at both the nucleotide and amino acid levels. As has been found for other triazine resistant psbA genes, the 'Triton' psbA contains an A to G nucleotide change which results in a serine to glycine amino acid substitution at position 264. The B. napus psbA gene also has a G insertion at position -9 resulting in a ribosome binding site sequence (AGGA) just before the initial methionine and suggesting that the entire open reading frame is translated. A large (72 bp) insertion is also found upstream of the B. napus psbA gene which resembles a similar insertion in the mustard psbA. The "uncloneable" nature of the entire gene is further investigated through reconstruction experiments and the implications discussed.

Sequences complementary to cellular deoxypolypyrimidines are localized in the 3' end of L-cell mRNA.

Many eukaryotic genomes have been shown to contain long pyrimidine tracts. In mouse L-cells, at least some of these pyrimidine tracts are transcribed and form a significant portion of the poly(A)+ RNA sequences. In this study the results from three different experimental methods indicate that the sequences complementary to polypyrimidines are localized to the 3' end of mRNA molecules. First, polypyrimidines reacted preferentially with the 3'-end fragments of mRNA generated by limited alkaline cleavage. Second, digestion of [3H]mRNA-polypyrimidine hybrids with RNase H released 3'-end fragments of mRNA which averaged only 250 nucleotides (NT) in length. Third, polypyrimidine tracts were isolated from cDNA which averaged only 200-250 NT extending from the 3' end of the corresponding mRNAs. These data suggest that the sequences transcribed from pyrimidine tracts are quite close to the 3' end of the cellular messages in which they occur, probably within the terminal untranslated region.

Alternariol, a dibenzopyrone mycotoxin of Alternaria spp., is a new photosensitizing and DNA cross-linking agent.

The mycotoxin alternariol (3,4',5-trihydroxy-6'-methyldibenzo [a] pyrone) but not alternariol monomethyl ether (3,4'-dihydroxy-5-methoxy-6'-methyldibenzo [a] pyrone) is phototoxic to Escherichia coli in the presence of near UV light (320-400 nm). The phototoxicity bioassays with a DNA repair-deficient mutant of E. coli suggested that DNA may be the molecular target for photo-induced toxicity of alternariol. Interactions between alternariol and double-stranded, supercoiled DNA suggest that alternariol interacts with DNA by intercalation. No DNA breakage was detected in this system; however, alternariol forms a complex and cross-links double-stranded DNA in near UV light. These results suggest that alternariol is a new phototoxic, DNA-intercalating agent and is a DNA cross-linking mycotoxin in near UV light.

An iron stress operon involved in photosynthetic electron transport in the marine cyanobacterium Synechococcus sp. PCC 7002.

The iron-stress-induced genes isiA and isiB have been cloned and sequenced from the marine unicellular cyanobacterium Synechococcus sp. PCC 7002. These genes code for a photosystem II chlorophyll-binding protein and flavodoxin respectively. The genes form a dicistronic operon that is transcriptionally activated under iron-stress conditions to produce an abundant monocistronic message containing isiA and a much less abundant dicistronic message that also contains isiB. The arrangement of these genes, their transcriptional control and the relative abundance of the monocistronic and dicistronic messages produced under iron stress parallels the pattern shown by the freshwater cyanobacterium Synechococcus sp. PCC 7942. The genes for the corresponding proteins found under iron-replete conditions, CP-43 and ferredoxin, have also been cloned and sequenced. Northern blot analysis indicates that both of these genes are constitutively expressed under both iron-stress and iron-replete conditions.

Effect of PCR conditions on the formation of heteroduplex and single-stranded DNA products in the amplification of bacterial ribosomal DNA spacer regions.

PCR amplifications of 16S/23S rDNA spacer regions were carried out from conserved 16S and 23S sequences for genomic DNA samples from strains representing 16 bacterial species (12 genera). Multiple products were produced containing conserved homologous sequences at the 3' and 5' ends, separated by highly variable internal spacer sequences. These products cross-hybridized forming heteroduplex DNA structures containing double-stranded ends surrounding an internal single-stranded loop. Single-stranded DNA was also produced in the amplification of rDNA spacer sequences. Fragments comprising the nonhomoduplex DNA components were identified by their susceptibility to removal by digestion with a single-stranded endonuclease. The relative formation of heteroduplex and single-stranded DNA was reduced by reaction conditions favoring primer/template annealing, for example, higher ionic strength, higher primer concentration, and lower annealing temperature, as well as by decreasing the number of amplification cycles. Heteroduplex and single-stranded DNA structures were also generated by denaturing and reannealing spacer amplification products in the absence of polymerase activity. Whereas heteroduplex and single-stranded DNA structures provide additional information that is helpful in distinguishing between species of bacteria that produce similar homoduplex products, the mobility of heteroduplex and single-stranded DNA structures DNA structures is extremely sensitive to electrophoretic conditions.

DNA-sequence organization in the genome of the domestic chicken (Gallus domesticus).

The sequence organization in the DNA of chicken (Gallus domesticus) was studied using hydroxyapatite-monitored reassociation kinetics. DNA 320-nucleotides long reassociates as though it is composed of three components, i.e., a very rapidly reacting fold-back fraction, a component composed of sequences repeated an average of 640 times in the genome, and a large unique fraction representing about 80% of the genome. The sizes of the fold back and repeated components increase only moderately with large increases in fragment size, indicating that these sequences are not extensively interspersed in the genome. Even at a fragment size of 4500 nucleotides, the unique component represents 68% of the DNA. Thus, the chicken genome is not organized in the short-period (Xenopus) interspersion pattern described for a large number of other organisms; rather, the DNA-sequence organization of this vertebrate bears more resemblance to the long-period interspersion pattern of Drosophila.

The localization of genes for ribosomal, cytochrome b6/f complex and photosystems I and II polypeptides on the chloroplast chromosome of Vicia faba.

Detailed studies of rearranged chloroplast genomes such as Vicia faba should give insights into the constraints governing the positional organization of the various gene clusters on the chloroplast chromosome. Seven polypeptide genes have already been mapped on the Vicia faba chloroplast genome (21, 22). In this report, ten additional chloroplast DNA-coded polypeptide genes have been mapped by heterologous hybridization. These genes include cytochrome b6 and subunit 4 of the cytochrome b6/f complex, the two P700 chlorophyll a apoproteins of photosystem I, the two chlorophyll a-binding proteins of photosystem II, cytochrome b559, the D2 polypeptide and two ribosomal proteins. The direction of transcription for five of these gene sequences has been established by utilizing 5' and 3' end specific gene probes. The genetic organization of the Vicia faba chloroplast genome was compared with the chloroplast maps of the standard conserved genome of spinach and the more rearranged genome of pea.

Photosystem II genes isiA, psbDI and psbC in Anabaena sp. PCC 7120: cloning, sequencing and the transcriptional regulation in iron-stressed and iron-repleted cells.

Under conditions of iron deprivation cyanobacteria produce flavodoxin to replace ferredoxin as the terminal electron acceptor of photosynthesis. In unicellular cyanobacteria, the gene for flavodoxin is the second open reading frame in a dicistronic operon whose transcription is tightly regulated by iron. The first gene, isiA, produces a protein that is very similar to CP43, a chlorophyll-binding, antenna protein of the photosystem II reaction center. In the filamentous, heterocystous cyanobacterium Anabaena sp. PCC 7120, isiA and the gene for flavodoxin are located in separate operons with independent promoters. In this paper, we report on the sequence of isiA and show that it is found in a monocistronic operon that is transcriptionally regulated to be expressed under iron stress but does not produce detectable transcripts under conditions of iron repletion. We also report on the sequence, organization and expression of the gene that codes for CP43, psbC. In Anabaena sp. PCC 7120, psbC has a genetic organization similar to that of other cyanobacteria and higher plants; the 5' end of psbC overlaps the 3' end of psbDI. Transcriptional analysis of the psbDC operon showed that it is constitutively expressed in both iron-repleted and iron-stressed conditions; however, a new monocistronic transcript was detected that contains psbC and is preferentially expressed under iron stress conditions.

Long pyrimidine tracts of L-cell DNA: localization to repeated DNA.

Like HeLa DNA, L-cell DNA contains a significant number of unexpectedly long pyrimidine tracts. 1.3% of the thymidine residues of L-cell DNA are found in these long polypyrimidine tracts. Analysis of L-cell DNA fractions with different rates of reassociation indicates that polypyrimidine tracts are associated with "repeated" DNA. Longer pyrimidine tracts appear to have a higher repetition frequency than shorter tracts, and some may be repeated as many as 5 x 10(4) times in L-cell DNA.

Relatedness of mouse satellite deoxyribonucleic acid to deoxyribonucleic acid of various Mus species.

Mouse (Mus musculus musculus) satellite DNA is able to reassociate with repeated DNA sequences of Mus caroli and Mus cervicolor, but low thermal stability of the products indicates significant differences between satellite and related DNAs of these two Mus species. There appear to be several satellite-related populations in M. caroli DNA, each of which forms hybrids of low thermal stability with repeated sequences of M. cervicolor DNA. DNAs from the subspecies Mus musculus molossinus and Mus musculus castaneus reassociate with mouse satellite to form hybrids of very high thermal stability, but the satellite content of M. m. musculus DNA is only about 60% that of M. m. musculus DNA. Reassociation of M. m. musculus nonrepeated DNA with M. m. molossinus DNA reveals no detectable differences between them; reassociation with M. caroli (or M. cervicolor) DNA yields a product whose melting temperature depression relative to homologous DNA is about 5 degrees .