Containment in industrial biotechnology within wastewater treatment plants.

Both physical and biological containment are considered to be essential parts in the risk analysis of industrial Good Industrial Large-Scale Practice (GILSP) processes using genetically modified organisms (GMOs). Biological containment of industrial microorganisms has become a more important issue since the introduction of recombinant DNA techniques. In the event of an accidental discharge in the production plant, a large amount of organisms could be released into the wastewater treatment (WWT) system. This WWT system should therefore be considered as a part of the containment. This study demonstrates both a hydrodynamic and a microbiological model for the containment aspects of industrial WWT plants. The models are verified by measurements using industrial hosts of GILSP GMOs at full scale. Both models describe the full-scale equipment accurately. The results are supplemented with microcosm studies on survival of GMOs in defined niches. It is shown that WWT plants can be considered as useful additional parts of the containment of microorganisms, in case of an accidental discharge. The effect of drainage of an enormous amount of microorganisms (several tons) through the WWT plant into the environment is shown to be comparable to the direct drainage of a small-scale fermenter. Microcosm experiments correlate well with the survival rates in the WWT and therefore can be of use to predict the behaviour of GMOs in this environment.

The gene for Spirodela oligorhiza chloroplast ribosomal protein homologous to E. coli ribosomal protein L16 is split by a large intron near its 5' end: structure and expression.

The nucleotide sequence of a Spirodela chloroplast DNA fragment, which directs the synthesis of a approximately 15 kD chloroplast ribosomal protein in an E. coli cell free system, has been determined. The deduced aminoacid sequence of the open reading frame shows extensive homology with E. coli ribosomal protein L16. Primer extension analysis, S1 nuclease mapping and nucleotide sequence analysis indicate that the chloroplast L16 gene (rpl16) is interrupted by a 1411 bp intron, which separates a short 5' exon from a large 3' exon. The shorter in vitro synthesized ribosomal protein results from an artificial initiation event at an internal ATG codon in the 3' exon. The sequences at the 5' and 3' splice sites of the intron are similar to consensus sequences described for other, class II intron containing, protein coding chloroplast genes. Northern hybridization experiments reveal 6 stable transcripts of rpl16 ranging from 500 b to greater than 4000 b. As determined by S1 nuclease mapping, the 3'-end of the smallest transcript maps exactly after the stem of a proposed termination signal. Finally, the implications of the finding of a cluster of several chloroplast ribosomal protein genes and possible polycistronic transcription of this chloroplast DNA region, are discussed in relation to the organization and expression of ribosomal protein genes found in the S10 operon of E. coli.

The genes encoding chloroplast ribosomal proteins S7 and S12 are located in the inverted repeat of Spirodela oligorhiza chloroplast DNA.

We have used a variety of methods to localize the genes for ribosomal proteins S7 and S12 on Spirodela chloroplast DNA. Heterologous hybridization with a rps12 gene specific probe from Euglena has revealed the presence of rps12 homologous sequences within the inverted repeat of Spirodela chloroplast DNA on the fragment BamHI-V. In the partial nucleotide sequence of this fragment, two regions of amino acid sequence homology to Euglena S12 can be identified, separated from each other by a 542 bp intron with conserved boundary sequences. As was found for Nicotiana S12, the Spirodela S12 coding regions are for 85 amino acids homologous (79%) to E. coli S12 (starting from residue 38 to the C-terminus). Likewise, we are unable to identify the 37 5' terminal codons of rps12 in Spirodela. The functionality of the Spirodela rps12 sequence is discussed. The rps7 gene is located adjacent to rps12. Chloroplast ribosomal protein C-S11 (homologous to S7) has been detected by immunoprecipitation with both a polyspecific anti 30S serum and an anti C-S11 serum, among the in vitro translation products of mRNAs selected by Spirodela chloroplast DNA fragments BamHI-V and BamHI-P. Since in a DNA dependent E. coli cell free system, only BamHI-V appears to be capable of synthesis of C-S11, it is concluded that rps7 is located entirely within BamHI-V and is transcribed into a mRNA which extends into BamHI-P. As determined by Northern hybridization experiments, rps7 is cotranscribed with rps12; a stable transcript of approx. 1100 b is detected in total cellular Spirodela RNA with either rps12 and rps7 gene specific probes. The rps12 probe additionally detects an approx. 600 b transcript, which presumably corresponds to the excised rps12 intron RNA. Finally we have examined the expression of both rps7 and rps12 during light induced chloroplast development by Northern blotting and by immunoblotting. It is shown, that the steady-state levels of neither chloroplast ribosomal protein transcripts, nor those of the chloroplast ribosomal proteins itself, change significantly during the greening process.

Induction of cytochrome P-450b,e-type isozymes by polychlorinated biphenyls in rat liver. Molecular cloning of induced mRNAs.

We have studied the induction of cytochrome P-450b,e-type antigen and mRNA by phenobarbital, 4,4'-dichlorobiphenyl and 2,4,5,2',4',5'-hexachlorobiphenyl in male Wistar rat liver. Chronic treatment of rats with 4,4'-dichlorobiphenyl leads to a relatively slow, 20-fold increase in the cytochrome P-450b,e-type antigen level and to an equivalent increase in the concentration of the corresponding mRNA. Treatment of rats with phenobarbital or 2,4,5,2',4',5'-hexachlorobiphenyl results in a faster and more pronounced increase of cytochrome P-450b,e-type antigen and mRNA levels. Analysis of clones from cDNA banks showed that two types of sequences are induced by phenobarbital corresponding to cytochrome P-450b and cytochrome P-450e respectively. 2,4,5,2',4',5'-Hexachlorobiphenyl appears to induce primarily a cytochrome P-450b-type sequence. The implications of these results for the study of the mechanism of induction are discussed.

Localization of three chloroplast ribosomal protein genes at the left junction of the large single copy region and the inverted repeat of Spirodela oligorhiza chloroplast DNA.

In order to determine the localization of ribosomal protein genes on the chloroplast genome of Spirodela, we have followed two different approaches: First, antisera were prepared against purified 30S, 50S and 70S chloroplast ribosomal proteins from Spinacia. These antisera react with about two third of the chloroplast ribosomal proteins as shown by protein blot and immunoprecipitation experiments. Recombinant plasmids carrying the Spirodela BamHI-G or PstI-I cpDNA fragment both direct the synthesis of a 15 kD chloroplast ribosomal protein in a DNA dependent E. coli cell free system. This was confirmed by molecular weight determination, immunoprecipitation and competition immunoprecipitation experiments. Second, heterologous hybridization with the rps19 gene probe from Nicotiana revealed the localization of this gene on the chloroplast DNA of Spirodela within the BamHI-G fragment at the left junction of the large single copy region and the inverted repeat. Furthermore we show that the recombinant plasmid carrying Nicotiana rps19 also directs the synthesis of another chloroplast ribosomal protein in an E. coli cell free system. The identity of this protein is discussed.

Repeated sequences on mitochondrial DNA ofSpirodela oligorhiza.

Mitochondrial DNA ofSpirodela oligorhiza (duck weed) was analyzed with restriction enzymes. The genome size appears to be at least 250 kbp. Four different PstI fragments were cloned. These four clones contain a sequence which is reiterated about 100-fold on theSpirodela mitochondrial DNA. Hybridization analysis showed that a similar sequence is present onZea mays mitochondrial DNA, although much less reiterated here. The presence of these reiterated sequences might contribute to the physical heterogeneity of plant mitochondrial DNA.

The nucleotide sequences of the genes coding for tRNAArgUCU, tRNAArgACG and tRNAAsnGUU on Spirodela oligorhiza chloroplast DNA.

The nucleotide sequences of the genes coding for tRNAArgUCU, tRNAArgACG and tRNAAsnGUU on chloroplast DNA of Spirodela oligorhiza have been determined. All three genes are expressed. 5' Proximal to these genes sequences are found homologous to prokaryotic promoter sequences, which might be involved as transcriptional start motifs.

Protein synthesis during chloroplast development in Spirodela oligorhiza. Coordinated synthesis of chloroplast-encoded and nuclear-encoded subunits of ATPase and ribulose-1,5-bisphosphate carboxylase.

We have studied qualitative and quantitative changes of several parameters during chloroplast development in Spirodela oligorhiza (duckweed). On a dry weight basis, the amount of protein increases from 2.5% (w/w) in dark-grown to 7.8% (w/w) in light-grown fronds. At the same time the amount of starch drops from 50% to 27% (w/w). Using an immunochemical quantification method we have found that during greening of etiolated plants the amount of all subunits of the ATPase complex per frond increases 10-fold, whereas the level of the subunits of ribulose-1,5-biphosphate carboxylase increases 50-fold. Cytochrome f was found to be present in dark-grown Spirodela and the amount of this polypeptide per frond increases about 30-fold. The concentration of a polypeptide that possibly represents a cytochrome b6 subunit increases about 10-fold upon greening. The molar ratio of the CF1-beta and CF1-gamma subunits of the ATPase complex varies over 2-3, while in all stages of chloroplast development studied the molar ratio of the carboxylase subunits is about 1. As these values are in agreement with the stoichiometrical amounts in the native protein complexes, we conclude that the synthesis of CF1-beta and CF1-gamma, as well as the synthesis of the large and small carboxylase subunits, are strictly coordinated during chloroplast biogenesis in Spirodela oligorhiza.

Transcription of ribosomal DNA in chloroplasts of Spirodela oligorhizaa.

The genes for the two large ribosomal RNAs (16S and 23S) and for the 4.5S rRNA in Spirodela oligorhiza chloroplast DNA are transcribed as one large, 7,000 nucleotides long precursor rRNA.Using S1-nuclease mapping, we have determined that the transcript ends 135 nucleotides 3' distal of the 4.5S rRNA gene. 5S rRNA therefore, is most likely transcribed separately.Northern blotting of chloroplast RNA with distinct probes derived from the rDNA region reveals RNAs, which can be described as intermediates in the processing of the large precursor. With these findings a pathway for the maturation of this precursor is proposed.

Isolation, characterization, phosphorylation and site of synthesis of Spinacia chloroplast ribosomal proteins.

We have characterized the ribosomal proteins from Spinacia chloroplasts using two-dimensional gel electrophoresis. The 30S and 50S subunits contain 23-25 and 36 ribosomal proteins, respectively. In contrast to prokaryotic ribosomes, chloroplast ribosomes contain at least one (and possibly two) phosphorylated ribosomal proteins. Isolated chloroplasts synthesize in the presence of ((35)S) labeled methionine and cysteine at least seven 30S and thirteen 50S ribosomal proteins which are assembled into (pre)ribosomes. This suggests that about one third of the chloroplast ribosomal proteins is encoded by the chloroplast DNA itself. The identity of several labeled proteins in the two-dimensional gel electrophoretic patterns which did not comigrate with stained chloroplast ribosomal proteins is discussed.

The nucleotide sequences of the regions flanking the genes coding for 23S, 16S and 4.5S ribosomal RNA on chloroplast DNA from Spirodela oligorhiza.

The nucleotide sequences of the flanking regions of the genes coding for Spirodela oligorhiza chloroplast ribosomal RNA's have been determined. We have compared these sequences to the corresponding ones in chloroplast DNA of other plants and of E. coli and find a striking sequential or structural homology. The region 5'-proximal to the gene coding for 16S rRNA contains a gene coding for tRNAval, which is transcribed from the same strand. In this area three prokaryotic promoter motifs are found: one located in front of the tRNAval gene and two in the intergenic space between this gene and the 16S rRNA gene. The middle one is used for the start of the transcription of the large ribosomal RNA precursor.

The nucleotide sequence of the 4.5S and 5S rRNA genes and flanking regions from Spirodela oligorhiza chloroplasts.

The base sequence of Spirodela oligorhiza chloroplast DNA coding for 4.5S and 5S ribosomal RNA, the flanking regions and the spacer between these two genes has been determined. We have compared these sequences with the corresponding ones in other higher plants. Besides a high degree of homology, some interesting differences are found.

Chloroplast DNAs of Spinacia, Petunia and Spirodela have a similar gene organization.

We have located the positions of the genes coding for the α, ß and ε subunits of the ATPase complex on Spirodela oligorhiza chloroplast DNA by means of heterologous hybridization with Spinacia cpDNA fragments.The overall cpDNA sequence organization of Petunia hybrida and Spirodela was compared. We hybridized well-characterized, cloned Spirodela cpDNA fragments with size fractionated Petunia cpDNA digested by Sall. It appears that the monocotyledonous Spirodela and the dicotyledonous Petunia cpDNA share a common sequence organization around their entire circumference. These observations, together with data reported in the literature, indicate a strikingly similar genetic organization of the chloroplast genome in widely divergent plants.

Molecular cloning and characterization of the chloroplast ribosomal RNA genes from Spirodela oligorhiza.

The organization of the chloroplast ribosomal RNA genes in Spirodela oligorhiza has been determined. We have therefore characterized two cloned BamHI fragments containing the genes for the large and small rRNA genes, and two PstI fragments, each containing one of the two large, invertedly repeated regions of chloroplast DNA. Characterization was performed by restriction endonuclease mapping, DNA/RNA hybridization and electronmicroscopic R-loop analysis.The results show that the rRNA genes are present in the order 16S, 23S, 4.5S and 5S. The 16S (1.5 kb) and 23S (2.9 kb) rRNA genes are separated by a spacer of 2.25 kb. There appears to be a close resemblance between the organization of the chloroplast rRNA genes in Spirodela oligorhiza and other higher plants so far examined.

RNA synthesis in isolated yeast mitochondria.

Isolated yeast mitochondria incorporate added UTP into RNA. Amongst the products formed are the two rRNAs, 4S RNA and several components presumed to be mRNAs. In omega+ strains (containing an intervening sequence in the 21S rRNA gene) besides mature 21S rRNA a transcript could be detected still containing nucleotide sequences transcribed from this intervening sequence. In omega- strains (not containing this intervening sequence) also a longer form of the 21S rRNA could be observed. These results suggest that isolated yeast mitochondria are capable of carrying out RNA synthesis and processing, including splicing.

Physical mapping of 4S RNA genes on chloroplast DNA of Spirodela oligorhiza.

We have determined the position of Spirodela oligorhiza chloroplast 4S RNA genes on the restriction fragment map of cp DNA, using purified in vitro[(32)P]-labeled 4S RNA. The overall organization of these genes is very similar to the organization of tRNAs on spinach cp DNA (Driesel et al. 1979).

Mutations affecting RNA splicing and the interaction of gene expression of the yeast mitochondrial loci cob and oxi-3.

In Saccharomyces cerevisiae strains KL14-4A and 777-3A, four intervening sequences of 1900 (l alpha beta), 1400 (l beta gamma), 1300 (l gamma delta) and 650 bp (l delta epsilon) separate the five coding sequences (alpha-epsilon) of the structural gene (cob) for cytochrome b. Its major transcript is an 18S RNA (2200 nucleotides) which is likely to be the functional mRNA. The lengths of a series of larger transcripts and their hybridization with probes specific for different intervening sequences are consistent with their being intermediates in a splicing process which generates 18S RNA from a giant primary transcript (greater than or equal to 7.5 kb) covering the whole cob region. There is no absolute order of splicing. The intervening sequence l alpha beta is excised in two stages. The first generates a stable 10S RNA, coded for by sequences immediately downstream of the 18S RNA coding segment alpha. The function of this RNA is unknown. Its excision is an early step in processing, whereas excision of the remainder of l alpha beta is a late event. We have studied four cytochrome b-deficient mutants. These map in intervening sequences and are splicing-defective. They accumulate 22S-28S RNAs which contain one or more intervening sequences. The l alpha beta mutants synthesize long, novel polypeptides, antigenically related to cytochrome b, possibly as a result of read-through into the intervening sequences. Several cob mutants also display alterations in their transcripts of oxi-3, the locus which codes for cytochrome c oxidase subunit I. This indicates that interactions between cob and oxi-3 exist at the level of RNA processing.

The organization of the genes for ribosomal RNA on mitochondrial DNA of Kluyveromyces lactis.

We have constructed a physical map of Kluyveromyces lactis mtDNA using the restriction enzymes HindII and HindIII. In contrast to Saccharomyces, the genes for the large and small ribosomal RNAs are much closer to each other, being separated by a maximal distance of 2,250 base pairs.