Repetitive-DNA elements are similarly distributed on Caenorhabditis elegans autosomes.

The positions of approximately 4,800 individual miniature inverted-repeat transposable element (MITE)-like repeats from four families were mapped on the Caenorhabditis elegans chromosomes. These families represent 1-2% of the total sequence of the organism. The four MITE families (Cele1, Cele2, Cele14, and Cele42) displayed distinct chromosomal distribution profiles. For example, the Cele14 MITEs were observed clustering near the ends of the autosomes. In contrast, the Cele2 MITEs displayed an even distribution through the central autosome domains, with no evidence for clustering at the ends. Both the number of elements and the distribution patterns of each family were conserved on all five C. elegans autosomes. The distribution profiles indicate chromosomal polarity and suggest that the current genetic and physical maps of chromosomes II, III, and X are inverted with respect to the other chromosomes. The degree of conservation of both the number and distribution of these elements on the five autosomes suggests a role in defining specific chromosomal domains.

Characterization of repetitive DNA elements in Arabidopsis.

We have applied computational methods to the available database and identified several families of repetitive DNA elements in the Arabidopsis thaliana genome. While some of the elements have features expected of either miniature inverted-repeat transposable elements (MITEs) or retrotransposons, the most abundant class of repetitive elements, the AthE1 family, is structurally related to neither. The AthE1 family members are defined by conserved 5' and 3' sequences, but these terminal sequences do not represent either inverted or direct repeats. AthE1 family members with greater than 98% identity are easily identified on different Arabidopsis chromosomes. Similar to nonautonomous DNA-based transposon families, the AthE1 family contains members in which the conserved terminal domains flank unrelated sequences. The primary utility of characterizing repetitive sequences is in defining, at least in part, the evolutionary architecture of specific Arabidopsis loci. The repetitive elements described here make up approximately 1% of the available Arabidopsis thaliana genomic sequence.

Rapid isolation of nuclei from carrot suspension culture cells using a BioNebulizer.

The BioNebulizer is an instrument that breaks cells and large molecules using the shearing forces created by laminar flow of high-pressure gas in the microcapillary channels generated by the instrument. Within 4 min, 90% of the carrot suspension culture cells that passed through the nebulizer were broken. Cytosol and organelles were released from the broken cells leaving cell wall ghosts. Nuclei were further purified by means of a discontinuous Percoll gradient. This method yielded an average of 2 x 10(5) nuclei from 2 g of suspension culture cells (approximately 2 x 10(6) cells). The isolated nuclei actively incorporated [8,5'-3H]-GTP into RNA.

Chloroplast RNA polymerase genes of Chlamydomonas reinhardtii exhibit an unusual structure and arrangement.

Nucleotide sequence analysis of a 17043 base-pair (bp) region of the Chlamydomonas reinhardtii plastome indicates the presence of three open reading frames (ORFs) similar to RNA polymerase subunit genes. Two, termed rpoB1 and rpoB2, are homologous to the 5'- and 3'-halves of the Escherichia coli beta subunit gene, respectively. A third, termed rpoC2, is similar to the 3'-half of the bacterial beta' subunit gene. These genes exhibit several unusual features: (1) all three represent chimeric structures in which RNA polymerase gene sequences are juxtaposed in-frame with long sequences of unknown identity; (2) unlike their counterparts in plants and eubacteria, rpoB1 and rpoB2 are separated from rpoC2 by a long (7 kilobase-pair, kbp) region containing genes unrelated to RNA polymerase; (3) DNA homologous to the 5' half of rpoC (termed rpoC1 in other species) is not present at the 5' end of rpoC2 and could not be detected in C. reinhardtii chloroplast DNA. RNA expression could not be detected for any of the RNA polymerase genes, suggesting that they are pseudogenes or genes expressed at stages of the C. reinhardtii life-cycle not investigated. The three genes are flanked by GC-rich repeat elements. We suggest that repeat DNA-mediated chloroplast recombination events may have contributed to their unusual arrangement.

Organization and structure of plastome psbF, psbL, petG and ORF712 genes in Chlamydomonas reinhardtii.

We have determined the nucleotide sequence of a 5159 base-pair (bp) region of the Chlamydomonas reinhardtii plastome containing three photoelectron transport genes, psbF, psbL and petG, and an unusual open reading frame, ORF712. The photosynthetic genes have an unprecedented arrangement, psbF and psbL are located in close proximity to petG, and are not grouped with two other genes of the cytochrome b559 locus, psbE and ORF42. ORF712, located adjacent to psbL, has homology at its 5'- and 3'-ends to the ribosomal protein rps3 gene, but contains a central 437 residue domain that lacks similarity to any other known sequence. These sequences add to the growing body of evidence that the chloroplast genome of C. reinhardtii has a significantly different gene arrangement to its counterpart in plants. The structure of ORF712 also provides another example of a phenomenon we have discovered with C. reinhardtii RNA polymerase genes (Fong and Surzycki 1992); namely, that the algal plastome contains chimeric genes in which reading frames with homology to known genes are juxtaposed in-frame with long coding regions of unknown identity.

Chloroplast and nuclear genomes of Chlamydomonas reinhardtii share homology with Escherichia coli genes for DNA replication, repair and transcription.

Most of the cpDNA genes studied to date are genes encoding elements of the photosynthetic apparatus and translational machinery. Much less is known about genes encoding the polypeptides involved in transcription, cpDNA replication, recombination and repair. The similarities between bacterial and some cpDNA genes were exploited to identify some of these chloroplast genes using bacterial probes. Probes derived from the Escherichia coli genes dnaA, recA, uvrC, transcriptional factor rho, and rpoC were used to search for homologous DNA sequences in chloroplast and nuclear genomes of Chlamydomonas reinhardtii. Regions homologous to all of these genes were located on the cpDNA physical map by probing restriction fragments of cpDNA with plasmid fragments containing these genes. Probing nuclear DNA with bacterial gene probes revealed DNA fragments homologous to dnaA and rpoC genes.

Screening recombinant clones containing sequences homologous to Escherichia coli genes using single-stranded bacteriophage vector.

Detection and isolation of Escherichia coli clones carrying vectors with foreign DNA sequences partially homologous to specific E. coli genes is difficult because denatured DNA in the host genome can hybridize with the probe. In this paper we present a procedure which simplifies this task by using bacteriophage M13 as the cloning vector. The procedure takes advantage of the secretory properties of the phage, as well as the property of nitrocellulose membrane to bind protein and single-stranded DNA but not double-stranded DNA. This procedure is shown to be effective in identifying E. coli clones containing sequences of Chlamydomonas reinhardtii chloroplast DNA that are homologous to the rpoC gene of E. coli. We suggest that this procedure can be used generally for rapid isolation of DNA sequences that are homologous to E. coli genes.

Both the chloroplast and nuclear genomes of Chlamydomonas reinhardi share homology with Escherichia coli genes for transcriptional and translational components.

Considerable DNA sequence homology can be detected between the Escherichia coli genes coding for translational and transcriptional components and both the chloroplast and nuclear genomes of Chlamydomonas reinhardi. Labeled chloroplast DNA was demonstrated to hybridize to DNA fragments of the transducing phages λfus3 and λspc2 that encode ribosomal proteins of the α and S10 operons. Further, chloroplast DNA probes hybridize to fragments of λrtf (d) 18 that encode the ß and ß' subunits of RNA polymerase. The regions homologous to the ribosomal protein and RNA polymerase genes were located on the chloroplast DNA physical map by probing restriction fragments of chloroplast DNA with phage or plasmid fragments carrying these E. coli genes. Probing nuclear DNA with bacterial gene probes revealed DNA fragments homologous to elongation factor and ribosomal protein genes. Most surprisingly, sequences homologous to the ß subunit of RNA polymerase were found not only in chloroplast DNA but in nuclear DNA as well.

Extensive sequence homology in the DNA coding for elongation factor Tu from Escherichia coli and the Chlamydomonas reinhardtii chloroplast.

Considerable DNA sequence homology can be detected between the Escherichia coli genes coding for translational components and Chlamydomonas reinhardtii chloroplast DNA. Labeled chloroplast DNA was found to hybridize to restriction fragments of the transducing phage lambda fus3 that code for elongation factor Tu. The chloroplast probe also reacts with fragments coding for ribosomal proteins carried by this phage. The region homologous to the elongation factor genes was located on the physical map of the chloroplast genome by probing restriction fragments of chloroplast DNA with cloned fragments, labeled in vitro, carrying the E. coli elongation factor Tu genes.

Location of binding sites for RNA polymerase II from wheat germ and from human placenta on adenovirus 2 DNA.

Electron microscopic visualization of binary complexes between eukaryotic RNA polymerases and Adenovirus 2 (Ad 2) DNA was used to locate specific binding sites for the enzymes. RNA polymerase II from human placenta binds to 10--16 distinct sites depending on the ratio of enzyme to DNA and the divalent cation present in the binding mixture. Wheat germ RNA polymerase binds to 12--14 strong binding sites and 2--3 weaker sites, all but one of which correspond to binding sites for the placental enzyme. At least six of the strong binding sites for both enzymes correspond to promoters known to be active in vivo. As a test of the two-state model for transcription initiation, we examined binding of wheat germ RNA polymerase II to Ad 2 DNA at 0 degrees and 37 degrees. The extent of binding was the same at the two temperatures and the distributions of binary complexes were virtually identical. This observation, in conjunction with results presented previously, is strong support for the existence of I and RS complexes in eukaryotic systems.

Transcription of adenovirus 2 DNA by human RNA polymerase II in vitro.

The interaction of Adenovirus 2 DNA and human placental RNA polymerase II in vitro satisfies criteria that suggest that at least some fraction of our purified polymerase preparations corresponds to prokaryotic holoenzyme and is able to initiate transcription at "true" promoters: (1) The purified enzyme forms highly stable complexes at specific sites on Ad 2 DNA; Kass = 1--2 X 10(12) M-1. (2) Transcription of Ad 2 DNA from pre-formed complexes with human RNA polymerase II is resistant to poly I. (3) Many of the stable-binding sites correspond to Ad 2 promoters known to be active in vivo. We also present evidence consistent with a two-state (I and RS) model (Chamberlin et al. 1976; Travers 1974) for the interaction of human RNA polymerase II with Ad 2 DNA. These experiments, which are similar to those described previously in studies of wheat germ RNA polymerase II (Seidman et al. 1979), indicate that the mechanisms of transcription inhibition and promoter site selection in eukaryotic and prokaryotic systems may be very similar.

E. coli ribosomal proteins are cross reactive with antibody prepared against Chlamydomonas reinhardi chloroplast ribosomal subunit.

Antisera prepared against purified Chlamydomonas reinhardi small chloroplast ribosomal subunit, judged homogenous by sucrose gradient velocity sedimentation and RNA gel electrophoresis was immunologically cross reactive with E. coli ribosomal proteins. The results of three different experimental approaches, namely Ouchterlony double diffusion, sucrose gradient velocity sedimentation and two dimensional crossed immunoelectrophoresis indicate that both E. coli ribosomal subunits and the chloroplast large ribosomal subunit contain proteins which show antigenic similarity to the chloroplast small ribosomal subunit proteins. However, cytoplasmic ribosomal subunits did not contain proteins which were cross reactive with immune antisera.

Interaction between wheat germ RNA polymerase II and adenovirus 2 DNA. Evidence for two types of stable binary complexes.

Transcription of Adenovirus 2 DNA (Ad 2 DNA) by wheat germ RNA polymerase II in vitro satisfies criteria that have been used to establish that Escherichia coli or coliphage transcription in vitro is initiated at true promoters. (1) Wheat germ RNA polymerase forms highly stable complexes at specific sites on Ad 2 DNA, with a Kassoc of (4--5) X 10(10) M-1. (2) Electron microscopic visualization of enzyme bound to Ad 2 DNA reveals the location of eight strong binding sites, at least five of which appear to correspond to promoters that have been identified in studies of Ad 2 transcription in vivo [Evans, R. M., Fraser, N., Ziff, E., Weber, J., Wilson, M., & Darnell, J.E. (1977) Cell 12, 733--739; Berk, A.J., & Sharp, P.A. (1977) Cell 12, 45--55; Weinmann, R., & Aiello, L. O. (1978) Proc. Natl. Acad. Sci. U.S.A. 75, 1662--1666]. (3) Transcription of Ad 2 DNA from preformed complexes with wheat germ polymerase is capable of escaping the action of rifamycin AF/013 and is relatively resistant to polyriboinosinic acid. In addition, our results are consistent with a two-state model for the interaction of wheat germ RNA polymerase with Ad 2 DNA, indicating that the mechansisms of transcription initiation and promoter-site selection in eucaryotes may be very similar to mechanisms elucidated in procaryotic systems.

Inactivation of E. coli RNA polymerase by polyriboinosinic acid: heterogeneity of RS complexes.

Polyriboinosinic acid (poly I) inhibits initiation of transcription by binary complexes formed between Adenovirus 2 DNA and E. coli RNA polymerase holoenzyme. In the presence of poly I, just as in the presence of rifampicin, initiation of transcription exhibits a sigmoidal dependence on the temperature at which the binary complexes are formed. This indicates that I (closed) complexes between Ad 2 DNA and RNA polymerase are rapidly inactivated by poly I, but that RS (open) complexes are relatively resistant. However, even among the RS complexes, at least two classes can be distinguished on the basis of the degree to which they are resistant to poly I: RS-1 complexes are somewhat sensitive to poly I (half-time of inactivation approximately 10 min) while RS-2 complexes are almost completely resistant to the inhibitor (half-time of inactivation approximately 10 h). For both types of RS complex, the degree of sensitivity to poly I is ionic strength-dependent.

Purification and characterization of a putative sigma factor from Chalamydomonas reinhardi.

Two proteins with sigma-like activity have been isolated from the alga, Chlamydomonas reinhardi. One protein, sigma 2, has been partially purified and appears to have a molecular weight of 51,000. The interaction of this protein with a heterologous (Escherichia coli) and homologous (Chlamydomonas, chloroplast rifampicin-sensitive) core RNA-polymerase (RNA nucleotidyltransferase, nucleosidetriphosphate: RNA nucleotidyltransferase, EC 2.7.7.6) was studied. Sigma 2 protein appears to stimulate the formation of open (rapid starting) binary complexes by both of the core enzymes. Stimulation of transcription by sigma 2 on chloroplast DNA was greater when Chlamydomonas core enzyme was used. Moreover, in vitro transcription on a variety of templates using RNA polymerases I and II from Chlamydomonas was not stimulated by this protein.

Characterization of the oxygenase activity in a mutant of Chlamydomonas reinhardi exhibiting altered ribulosebisphosphate carboxylase.

A previously described Mendelian mutant of Chlamydomonas reinhardi, ac i72, exhibiting altered ribulosebisphosphate carboxylase activity and unable to grow on minimal medium is examined for changes in ribulosebisphosphate oxygenase activity. The ribulosebisphosphate oxygenase activity of the enzyme purified from both wild type and ac i72 is compared over a pH range from 7.0 to 9.5. Both enzymes exhibit maximum activity at pH 9.0. However, the ac i72 enzyme is twice as active as the wild type enzyme at a physiological pH of 7.0. The studies in vivo of the products of CO2 fixation of ac i72 and wild type cells in the presence of high and low O2 concentration shows that due to a lower level of carboxylation, the ac i72 cells fix CO2 at half the rate of wild type cells. In ac i72, 24% of the photosynthetically fixed 14C is channelled into the water-soluble fraction as opposed to 6% in wild type. Thin-layer chromatography of the water-soluble fraction showed extensive accumulation of components of the glycolate pathway in ac i72 as compared to wild type. This indicates that the oxygenase activity of the enzyme prevails in ac i72 in vivo. Since a high concentration of glycolate is toxic to cells of C. reinhardi, the high oxygenase activity of ac i72 provides an explanation for the inability of ac i72 to grow phototrophically even though its rate of CO2 fixation is half that of wild type. This toxicity to glycolate is overcome by growth under amber illumination or low O2 concentration.

I.n vitro transcription of adenovirus 2 DNA. I. Characterization of promoters for E. coli RNA polymerase.

E coli RNA polymerase holoenzyme is able to recognize transcription initiation sites on Adenovirus 2 DNA that are functionally indistinguishable from promoters for the enzyme on phage DNAs. The complexes formed between the polymerase and the DNA at these sites can exist in two states-either as I (initiation) complexes, from which rapid RNA chain initiation is not possible, or as RS (rapid starting) "rifampicin resistant" complexes, from which rapid RNA chain initiation can occur. When transcription is limited to that initiated from stable, rifmapicin-resistant pre-initiation complexes, initiation is strictly dependent on the presence of sigma factor; in addition, the frequency of initiation exhibits sigmoidal dependence on the temperature at which pre-initiation complexes are allowed to form, with a transition temperature of 26-28 degrees C. The average half-time for initiation of RNA chains from sites on Ad 2 DNA is shown to be comparable to half-times for initiation of RNA chains from promoters on T7 and lambda DNAs. At saturating levels of enzyme, the half-times are 0.6, 0.9, and 1.6 sec for lambda b2, Ad 2 and T7 DNAs, respectively. The existence of efficient, phage-like promoters for E coli RNA polymerase on Ad 2 DNA suggests to us that such promoters may be closely related functionally and spatially to promoters for mammalian RNA polymerases.

In vitro transcription of adenovirus 2 DNA. II. Quantification and localization of promoters for E. coli RNA polymerase.

We estimate that E. coli RNA polymerase is able to form stable, rifampicin-resistant, pre-intiation complexes with Adenovirus 2 DNA at three to six binding sites. The number of RNA chains initiated from such complexes has been determined form the incorporation of gamma-32P-ATP and -GTP at two rifampicin concentrations (7 mug/ml and 24 mug/ml) and after pre-incubation at either 25 or 37 degrees C. The total number of RNA chains initiated ranges from 2.6 per Ad 2 DNA molecule at a rifampicin concentration of 24 mug/ml and pre-incubation temperature of 25 degrees C, to 5.4 per Ad 2 DNA molecule at a rifampicin concentration of 7 mug/ml and pre-incubation temperature of 37 degrees C. Efficient initiation with GTP occurs only after pre-incubation at 37 degrees C whereas initiation with ATP is equally as efficient at either pre-incubation temperature. Promoters for initiation with ATP have been localized to the leftmost 58% of the Ad 2 DNA molecule, defined by the EcoR.RI restriction endonuclease fragment A; promoters for initiation with GTP are located on the remaining 42% of the Ad2 DNA molecule. It is likely that on Adenovirus 2 DNA each RNA chain is initiated from a unique binding site which constitutes a seperate promoter for E. coli RNA polymerase.

A mutant strain of Chlamydomonas reinhardi exhibiting altered ribulosebisphosphate carboxylase.

A mutant, ac i72, of Chlamydomonas reinhardi possessing an altered ribulosebisphosphate carboxylase and unable to grow on minimal medium has been isolated and characterized. Comparison of ribulosebisphosphate carboxylase purified from both wild type and ac i72 strains is given. The enzyme from ac i72 shows alterations in several characteristics: (a) the specific activity is reduced to 35% that of wild type, (b) the V for both substrates is reduced 3-6 fold, (c) the Mg2+ requirement for maximal activity is 3 times greater, (d) the inhibitory effect of Cl- is greater, and (e) the isoelectric point is changed (6.0 for wild type and 5.8 for ac i72). However, the ribulosebisphosphate carboxylase from ac i72 is identical to that from wild type with respect to pH requirement, temperature sensitivity, subunit structure, and sedimentation characteristic. Other photosynthetic properties of wild type and ac i72 cells were also compared. CO2 fixation in ac i72 in vivo is reduced proportionally to the reduction in activity of the enzyme, but the level of O2 evolution is the same as in wild-type cells. Photosynthetic electron transport, 70-S ribosome content, and chlorophyll content are unaltered in ac i72. The chloroplast ultrastructure of ac i72 cells is distinctly different from that of wild-type cells. The inheritance of the mutation is Mendelian.