Biomedical subjects

H Kössel

Publications and source records attributed to H Kössel.

At least 37 records · Page 2Linked to original sources

PubMed1995

Complete sequence of the maize chloroplast genome: gene content, hotspots of divergence and fine tuning of genetic information by transcript editing.

The nucleotide sequence of the chloroplast (cp) DNA from maize (Zea mays) has been completed. The circular double-stranded DNA, which consists of 140,387 base-pairs, contains a pair of inverted repeat regions (IRA and IRB) with 22,748 base-pairs each, which are separated by a small and a large single copy region (SSC and LSC) of 12,536 and 82,355 base-pairs, respectively. The gene content and the relative positions of a total of 104 genes (70 peptide-encoding genes, 30 tRNA genes and four rRNA genes) are identical with the chloroplast DNA of the closely related species rice (Oryza sativa). A detailed analysis of the two graminean plastomes allows the identification of hotspots of divergence which predominate in one region containing a cluster of tRNA genes and in two regions containing degenerated reading frames. One of these length differences is thought to reflect a gene transfer event from the plastome to the nucleus, which is followed by progressive degradation of the respective chloroplast gene resulting in gene fragments. The other divergent plastome region seems to be due to the complete loss of a plastid gene and its functional substitution by a nuclear encoded eukaryotic homologue. The rate of neutral nucleotide substitutions is significantly reduced for protein coding genes located in the inverted repeat regions. This indicates that the existence of inverted repeat regions confers increased genetic stability of the genes positioned in these regions as compared to genes located in the two single copy regions. Editing events cause the primary structures of several transcripts to deviate from the corresponding genomic sequences by C to U transitions. The unambiguous deduction of amino acid sequences from the nucleotide sequences of the corresponding genes is, therefore, not possible. A survey of the 25 editing positions identified in 13 different transcripts of the maize plastome shows that representatives of all protein coding gene classes are subject to editing. A strong bias exists for the second codon position and for certain codon transitions. Based on the number and the codon transition types, and taking into account the frequency of putative editing sites in all peptide encoding genes and unidentified reading frames, a total number of only few more than the experimentally verified 25 editing sites encoded in the maize plastome is estimated. This corresponds to 0.13% of amino acid positions which cannot be derived from the corresponding codons present in the corresponding genes.

Base Sequence↗

PubMed1995

Editing of the chloroplast ndhB encoded transcript shows divergence between closely related members of the grass family (Poaceae).

The ndhB-encoded transcript from barley chloroplasts deviates from the genomic ndhB sequence by nine C-to-U transitions, which is the maximum number of editing events for a chloroplast mRNA reported so far. Comparison with ndhB transcripts from other chloroplast species shows that six of the nine editing sites observed in barley are structurally and functionally conserved in maize, rice and tobacco. The remaining three sites, however, show divergent patterns of conservation even within the three members of the grass family. The conservation of two of these sites in tobacco but not in the closely related graminean species suggests that divergence of the ndhB editing sites is caused by the loss of preexisting editing sites rather than by gain of new sites.

Amino Acid Sequence↗

PubMed1995

Structural and functional analysis of plastid genomes from parasitic plants: loss of an intron within the genus Cuscuta.

The gene cluster rps12/rps7/psi ndhB of the plastome from the holoparasitic plant Cuscuta europaea has been analysed at the nucleotide level. A comparison with the homologous region of the plastome from the closely related parasite Cuscuta reflexa reveals a complete loss of the cis-spliced intron of the rps12 gene in addition to a drastic size reduction of the ndhB pseudogene. It is demonstrated by RT-PCR analysis that the entire gene cluster is transcribed in the form of a multicistronic transcript which also includes the sequences encoded by the ndhB pseudogene. A cDNA containing the correctly transpliced exon 1 of the rps12 transcript can also be amplified. This shows that trans-splicing of the rps12 transcript persists in the plastids of the holoparasite despite the loss of the cis-spliced intron and the loss of many other gene functions. The rps12 and rps7 genes, therefore, still appear to code for functional ribosomal proteins CS12 and CS7, respectively. The conservation of apparently intact ribosomal-protein genes from which correctly processed transcripts are produced is taken as evidence that the translational apparatus of the plastids is still functional and necessary for the expression of the genes remaining in the reduced plastome of a parasitic plant.

Amino Acid Sequence↗

PubMed1995

[Extracorporeal membrane oxygenation of the newborn infant--an example of interdisciplinary project management].

In the last three years an interdisciplinary ECMO (extracorporeal membrane oxygenation) team has been set up at the Free University of Berlin in the Steglitz clinic. With this form of management, newborns only a few hours or days old who present with pulmonary failure can temporarily be managed with extracorporeal oxygenation until normal lung function is restored. The set up of this ECMO team, starting with experimental training in animal models, to the bedside clinical use is discussed. The following presentation of the first four cases managed here with ECMO aims to clarify the problems which may be encountered as well as amplifying its use as a life-saving measure.

Asphyxia Neonatorum↗

PubMed1995

The influence of lung injury due to mechanical ventilation on the initiation of ECMO.

Before the entry criteria for extracorporeal membrane oxygenation (ECMO) are met, newborns may require aggressive mechanical ventilation which may result in lung injury. The question arises whether the presence of a pneumothorax in these infants plays a role in the prognosis. Of the 21 newborns transferred to our hospital for ECMO, 8 were treated with ECMO. 9 of the 21 newborns developed a pneumothorax with conventional ventilation and 6 of these 9 newborns subsequently required ECMO. Infants who developed a pneumothorax but did not meet ECMO criteria and remained in the oxygenation index (OI) range between 25 and 40 for more than 2 days had a poorer prognosis. If adequate oxygenation cannot be attained with acceptable mechanical ventilation and a more aggressive ventilation results in a pneumothorax, ECMO should be considered even if the oxygenation index is below 40.

Blood Gas Analysis↗

PubMed1994

Introduction of a heterologous editing site into the tobacco plastid genome: the lack of RNA editing leads to a mutant phenotype.

The psbF mRNA is edited in spinach plastids by a C to U conversion, changing a serine to a conserved phenylalanine codon. In tobacco at this position a phenylalanine codon is present at the DNA level, and the psbF mRNA here is not edited. To test if the psbF editing capacity is evolutionarily conserved, the tobacco psbF gene was modified to match the corresponding spinach sequence. The endogenous tobacco gene was replaced with the modified copy using biolistic transformation. We report here that the heterologous editing site remains unmodified in transplastomic tobacco plants. The lack of editing is associated with slower growth, lowered chlorophyll content and high chlorophyll fluorescence, a phenotype characteristic of photosynthetic mutants. This finding confirms that the editing of the psbF mRNA is an essential processing step for protein function and thus provides direct proof for the biological significance of plant organellar RNA editing. Given that a mutant phenotype is associated with the lack of editing, it seems likely that the evolutionary loss of the site-specific capacity for psbF editing was preceded by the mutation that eliminated the editing requirement.

Amino Acid Sequence↗

PubMed1994

The role of RNA editing in conservation of start codons in chloroplast genomes.

Open reading frames (ORFs), encoded by the plastid genomes of tobacco, liverwort, rice and maize were aligned with a view to studying the conservation of translational start and stop codons created by RNA editing of homologous genes. It became evident that most of the homologous ORFs have conserved translation start and stop signals at the gene level. However, some of the ORFs show differences with respect to extensions of their 3' and 5' terminal regions. For example, the proposed N-termini of the ndhD-encoded peptides from different plant species are very variable in length and amino-acid composition. Sequence analysis of ndhD and the corresponding cDNA shows that editing of an ACG triplet in tobacco, spinach and snapdragon leads to the creation of an AUG codon, corresponding to the start codon in other species. Conservation of translational start codons of plastome-encoded genes can, therefore, be achieved by editing of transcripts, and the definition of plastome-encoded ORFs must take potential editing events into consideration.

Amino Acid Sequence↗

PubMed1994

Complete RNA editing of unspliced and dicistronic transcripts of the intron-containing reading frame IRF170 from maize chloroplasts.

The maize plastome harbors within the rps4-rps14 gene cluster the reading frame IRF170, which is interrupted by two introns. Although the function of the encoded peptide of 170 amino acids is not known, the conservation of IRF170 homologs in other plastomes is a strong indication that IRF170 is a functional gene. Amplification and sequence analyses of IRF170 specific cDNAs reveals two C-to-U editing events occurring within each of the first two exons. This situation allows an analysis of the temporal order between editing and splicing of a chloroplast transcript. By using intron-specific primer combinations, cDNAs derived from partially or even unspliced IRF170 transcripts could be amplified which in all cases showed complete editing. Complete editing was also observed with a cDNA derived from a transcript in which the proximal rps4 and the 5' half of IRF170-encoded sequences were still linked. This demonstrates that editing of the IRF170 transcript is an early processing step preceding both splicing and cleavage to monocistronic mRNA.

Amino Acid Sequence↗

PubMed1994

RNA editing of tobacco petB mRNAs occurs both in chloroplasts and non-photosynthetic proplastids.

We found an RNA editing site in the protein coding region of tobacco (Nicotiana tabacum) petB transcripts. This editing (CCA to CUA) leads to an amino acid conversion from proline to leucine. It is observed not only in chloroplasts isolated from tobacco leaves but also in non-photosynthetic proplastids isolated from a tobacco cell culture. Also unspliced pre-mRNA shows complete editing. The editing site is the same as that recently observed in the maize petB transcripts which restores the codon for a highly conserved leucine residue, suggesting that RNA editing at this site is critical for the protein (cytochrome b6) function.

Base Sequence↗

PubMed1994

Inefficient rpl2 splicing in barley mutants with ribosome-deficient plastids.

Analysis of transcript accumulation and splicing in plastids of four nuclear mutants of barley revealed that the ribosomal protein L2 (rpl2) gene transcripts containing a group II intron remained entirely unspliced, whereas the intron of the ribosomal protein L16 (rpl16) gene (linked with the rpl2 gene in the same operon) was removed in the mutant plastids. Also, the transcripts of other genes containing group II introns (ribosomal protein S16 gene, rps16; NADH dehydrogenase ND2 gene, ndhB; cytochrome f gene, petD; and intron-containing reading frame 170, irf170) and of the tRNA for leucine, trnL (UAA), possessing the only chloroplast group I intron, were found to be spliced. The mutants used in this investigation are considered to be nonallelic; this excludes the possibility that a single nuclear gene is responsible for the impaired splicing of rpl2 transcripts. The mutants, however, have a severe deficiency in chloroplast ribosomes in common; this deficiency is evident from the lack of the essential ribosomal protein L2 and from an extremely low steady state level of plastid rRNAs. From these results, we conclude that a functioning translational apparatus of the organelle is a prerequisite for splicing of the chloroplast rpl2 class II intron but not for splicing of at least five other group II intron-containing transcripts. This provides genetic evidence for a chloroplast DNA-encoded component (e.g., a maturase) involved in the splicing of rpl2 pre-mRNA.

Base Sequence↗

PubMed1993

Editing of the chloroplast rpoB transcript is independent of chloroplast translation and shows different patterns in barley and maize.

Sequence analysis of amplified cDNAs derived from the maize chloroplast rpoB transcript which encodes the beta subunit of a chloroplast specific, DNA dependent RNA polymerase reveals four C-to-U editing sites clustered within 150 nucleotides of the 5' terminal region of the rpoB message. These newly identified editing sites confirm the bias of chloroplast editing for certain codon transitions and for second codon positions which both appear suggestive for an involvement of the translational apparatus in the editing process. This supposition prompted us to investigate editing of the rpoB transcript from ribosome deficient, and hence protein synthesis deficient, plastids of the barley mutant albostrians. In this mutant editing is, however, not impaired at any of the editing sites functional in the barley wild type rpoB transcript. This demonstrates that chloroplast editing is neither linked to nor dependent on the chloroplast translational apparatus. As a further consequence any peptide components required for chloroplast editing must be encoded in the nuclear genome. In spite of strong sequence conservation only three of the four editing sites identified in the maize rpoB transcript are functional in barley. This indicates that sequences surrounding an editing site alone are not sufficient as determinants for the editing process in chloroplasts, but that trans-acting templates carrying the editing information for each individual site may also be required.

Amino Acid Sequence↗

PubMed1993

Tissue- and stage-specific modulation of RNA editing of the psbF and psbL transcript from spinach plastids--a new regulatory mechanism?

The psbE operon of spinach chloroplasts, which includes the genes psbE, psbF, psbL and psbJ, encodes two RNA editing sites. One site corresponds to the initiation codon of the psbL transcript, as has been described earlier for the homologous transcript from tobacco, while at a second editing site, newly reported here, an internal phenylalanine codon of the psbF transcript is restored. Both these sites were investigated with respect to the extent of editing in spinach plastids at various developmental stages. The apparent existence of only completely edited transcripts in etioplasts and chloroplasts, indicates that light-induced processes are not acting as determinants in eliciting the editing process. Reduced editing is, however, observed in the psbF and psbL transcript from seeds and roots. This finding suggests that the RNA editing process is differentially down-regulated in leucoplasts and proplastids and that editing may, therefore, function as a regulatory device in plastid gene expression.

Amino Acid Sequence↗

PubMed1993

RNA editing in maize chloroplasts is a processing step independent of splicing and cleavage to monocistronic mRNAs.

The psbB operon contained in the plastomes of higher plants consists of the genes psbB, psbH, petB and petD. The primary transcript of this operon is subject to a series of processing steps which include cleavages resulting in four monocistronic mRNAs and splicing of the petB and petD transcripts. A search for editing sites within the two latter transcripts from maize led us to the detection of one editing site within the petB coding region which is conserved at the DNA level in other graminean species and in tobacco. This shows that editing must be considered as an additional processing step of the psbB operon encoded primary transcript. As is evident from cDNA sequences derived from the dicistronic and/or unspliced petB/D transcripts which are completely edited, editing is an early step of mRNA processing which precedes both splicing and cleavage to the monocistronic mRNAs and which must, therefore, be independent of the latter two steps. This conclusion is confirmed by a similar observation with the editing site of the rpl2 transcript which is contained in the polycistronic transcript of the rpoA operon, although here only partial editing is observed for the unspliced dicistronic rpl23/rpl2 transcript.

Animals↗

PubMed1992

Identification of editing positions in the ndhB transcript from maize chloroplasts reveals sequence similarities between editing sites of chloroplasts and plant mitochondria.

A comparison of the nucleotide sequences from genomic DNA and cDNA of the ndhB gene from maize chloroplasts shows that the ndhB transcript is edited by C-to-U transitions at six positions which appear to exist as editing sites also in the chloroplast ndhB genes from rice and tobacco but not from liverwort. In order to identify possible sequence determinants necessary for editing, the sequences surrounding the newly identified ndhB and previously identified ndhA editing sites were compared with each other and with editing sites observed in plant mitochondrial transcripts. Among the chloroplast editing sites two closely positioned ndhB sites show similarity by sharing a common octanucleotide. The existence of the identical octanucleotide in the ndhJ gene whose transcript is not edited at the respective position, shows, however, that this octanucleotide is not sufficient to elicit the editing process. On the other hand, several of the chloroplast editing sites show sequence similarities with certain sets of consensus sequences reported earlier for editing sites of plant mitochondria. This supports the view that the editing processes of both plant organelles share common components and/or mechanistic steps and that the consensus sequences are part of the determinants necessary for editing.

Amino Acid Sequence↗

PubMed1992

RNA editing in tobacco chloroplasts leads to the formation of a translatable psbL mRNA by a C to U substitution within the initiation codon.

The psbL gene which codes for a 38 amino acid peptide of photosystem II, together with the photosynthetic genes psbE and psbF, is contained in a conserved position of many species of higher plant plastomes. The alignment of the psbL nucleotide sequences from ten species shows strong conservation, which is indicative of a functional gene. The tobacco and spinach psbL genes have, however, an ACG codon instead of the initiator ATG codon observed in the homologous position of the other eight species. Evidence is presented that in tobacco chloroplasts a translatable psbL mRNA containing an AUG initiator codon is formed by a C to U editing of the ACG codon. This observation, following the previously reported editing of an rpl2 gene in maize chloroplasts, underlines a more widespread occurrence of this type of posttranscriptional mRNA modification and demonstrates its presence in a dicotyledon plant.

Base Sequence↗

PubMed1992

Isolation and characterization of a gene encoding a chlorophyll a/b-binding protein from mustard and the targeting of the encoded protein to the thylakoid membrane of pea chloroplasts in vitro.

Three independent clones carrying a mustard gene coding for the chlorophyll a/b-binding protein were isolated by screening a genomic library of mustard with a heterologous cDNA probe from pea. All of them encode the same CAB gene, which, as shown by sequence analysis and comparison with published CAB sequences, belongs to the family of type I PSII CAB genes, encoding a precursor protein of 266 amino acids. Several conserved sequence motifs are observed in the 5' and 3' non-coding region of the gene. The putative transcription start site could be localized to 60 bp upstream of SA-CAB1 initiator codon by S1 mapping. Plasmids were constructed which allow in vitro transcription and translation of the whole chlorophyll a/b-binding protein and of truncated species which lack increasing portions of the C-terminus. Whereas the in vitro import into pea chloroplasts is not affected by these C-terminal deletions, targeting to the thylakoid membrane is abolished by the removal of the C-terminal helical domain. Accordingly, the 54 amino acids which contain the C-terminal membrane-spanning helix and flanking regions is an essential component of the thylakoid targeting signal.

Amino Acid Sequence↗

PubMed1992

Internal editing of the maize chloroplast ndhA transcript restores codons for conserved amino acids.

The NADH dehydrogenase subunit A (ndhA) gene from maize chloroplasts encodes a highly conserved peptide, which at several positions could be restored to consensus sequences by potential C-to-U editing of the codons involved. This gene was, therefore, chosen for analysis of its mRNA sequence in the form of amplified cDNA. A comparison of this cDNA sequence with the plastome-encoded ndhA sequence reveals four C-to-U editing sites, thereby demonstrating as a novel finding that chloroplast editing can also affect internal mRNA positions. All the edited codons restore amino acids that are conserved in the ndhA-encoded peptides of other chloroplast species. Alignment with homologous mitochondrial NADH-ubiquinone reductase subunit 1 (nad1) sequences of plant and even nonplant species shows that two of the editing positions restore universally conserved amino acids and that one editing site is even shared with nad1 mRNA of plant mitochondria. No editing sites could be detected in the cDNA derived from transcripts of the maize chloroplast RNA polymerase alpha-subunit (rpoA) gene.

Amino Acid Sequence↗

PubMed1991

Editing of a chloroplast mRNA by creation of an initiation codon.

Primary mRNA transcripts in several systems are edited by single base substitutions, small deletions or insertions to yield functional messenger RNA species. Mitochondrial mRNAs in particular, including those from plants, seem to be the subject of extensive editing, unlike mRNAs encoded by chloroplast DNA, for which the prediction of amino-acid sequence from the corresponding gene sequence is generally unambiguous. Occasionally, however, an ACG codon appears at the 5' terminus of chloroplast genes, where the initiation codon ATG would be expected. Here we present evidence for a C----U editing that is responsible for the conversion of the ACG codon to an AUG initiation codon in the mRNA transcript from the rpl2 gene of the maize plastome, showing that mRNA editing can also occur in chloroplasts.

Amino Acid Sequence↗