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Biomedical subjects

D A Christopher

Publications and source records attributed to D A Christopher.

30 records · Page 2Linked to original sources
PubMed1995

The effect of refraction and assumed speeds of sound in tissue and blood on Doppler ultrasound blood velocity measurements.

The combined effect of three assumptions relating to refraction, the speed of sound in tissue and the speed of sound in blood on the accuracy of Doppler ultrasound blood velocity measurements has been investigated. A theoretical relationship giving the net velocity measurement error introduced by these three assumptions has derived using a model in which tissue and blood layers are separated by straight, parallel boundaries. This net error is dependent on the assumed and actual speed of sound in tissue, the assumed speed of sound in blood and the Doppler angle, but is effectively independent of the actual speed of sound in blood. For clinical blood velocity measurements, the net error is estimated to be as much as an 8% overestimation of the actual velocity, higher than previously predicted for any of the factors individually. The relationship also predicts a net velocity measurement error in experimental flow systems and string phantoms which is dependent on the speed of sound in the liquid bath. A water bath at room temperature will give an overestimation of approximately 2%. Experimental investigations using conventional and modified string phantoms and a 5-MHz linear phased array system support these conclusions. The effect of perturbing the layers from their parallel orientation has also been considered theoretically and has provided additional support for the above conclusions. These results may help more accurate Doppler velocity measurements in both experimental and clinical settings.

Blood↗
PubMed1994

Separate photosensory pathways co-regulate blue light/ultraviolet-A-activated psbD-psbC transcription and light-induced D2 and CP43 degradation in barley (Hordeum vulgare) chloroplasts.

We studied the effects of spectral quality and fluence on the expression of several chloroplast-encoded photosynthesis genes and on the stability of their protein products in barley (Hordeum vulgare). During light-dependent chloroplast maturation, mRNA levels for psbD-psbC and psbA were maintained at higher levels compared with mRNAs encoding proteins for other photosynthesis functions (atpB, rbcL). Maintenance of psbD-psbC mRNA levels was accounted for by differential activation of the psbD-psbC light-responsive promoter by high-irradiance blue light and, secondarily, ultraviolet A (UV-A) radiation. Promoter activation was fluence dependent and required continuous illumination for 2 h at threshold fluences of 1.3 (blue light), 7.5 (white light), or 10 (UV-A) mumol m-2 s-1. From immunoblot analysis experiments, we showed that the psbD-psbC gene products D2 and CP43 undergo light-mediated turnover similar to light-labile D1. Other photosynthesis proteins such as the beta subunit of ATP synthase and the large subunit of ribulose-1,5-bisphosphate carboxylase were relatively stable. In the absence of protein synthesis, D2 degradation paralleled the degradation of D1 (relative half-lives, 9.5-10 h). CP43 decay was about half of D2 and D1 decay. In contrast with activation of the light-responsive promoter, the fluence-dependent degradation of D1, D2, and CP43 required 50- to 100-fold higher fluences of photosynthetically active white, red, blue, or UV-A irradiation. We interpret the different fluence and wavelength requirements to indicate that separate photosensory systems regulate activation of psbD-psbC transcription and turnover of D1, D2, and CP43. We propose that a blue light/UV-A photosensory pathway activates the psbD-psbC light-responsive promoter, differentially maintaining the capacity of mature chloroplasts to synthesize D2 and CP43, which are damaged and turned over in illuminated plants.

Chloroplasts↗
PubMed1993

Direct evidence for selective modulation of psbA, rpoA, rbcL and 16S RNA stability during barley chloroplast development.

The turnover of RNAs encoded by seven different barley chloroplast genes was analyzed after treatment of barley shoots with tagetitoxin, a selective inhibitor of chloroplast transcription. Changes in RNA stability were examined during chloroplast development using basal and apical leaf sections of 4.5-day-old dark-grown seedlings and apical leaf sections of 4.0-day-old dark-grown seedlings which had been illuminated for 12 h. Of the RNAs examined, a 2.6 kb unspliced precursor of tRNA(lys) exhibited the shortest half-life, which was estimated to be 3 h. The 16S rRNA and psbA mRNA had the longest estimated half-lives, which were greater than 40 h. Among mRNAs, half-lives were estimated to range from 6 h for psaA mRNA, to over 40 h for psbA mRNA. Therefore, barley chloroplast mRNAs have long half-lives relative to bacterial mRNAs. The stability of atpB mRNA and the unspliced precursor of tRNA-lys was not altered during chloroplast development, while the stability of psaA mRNA decreased 2-fold. In contrast, the stability of the 16S rRNA and mRNAs for rpoA, psbA and rbcL increased during chloroplast development. The stability of 16S rRNA increased markedly during chloroplast development in the dark and this increase was maintained in illuminated seedlings. The stability of rbcL mRNA increased 2.5-fold during chloroplast development in the dark, and then decreased 2-fold in chloroplasts of light-grown plants. The initial increase in rpoA and psbA mRNA stability was also light-independent, with total increases in stability of at least 5-fold. In the case of rpoA, the stability of 2 of the 13 polycistronic rpoA transcripts that were detected in dark-grown plants was selectively increased during chloroplast development. In conclusion, the stability of some transcripts is selectively increased and further modulated during chloroplast development in barley. We propose that the selective stabilization of chloroplast mRNA, which occurred independent of light, is an indication that non-light regulated developmental signals are involved in barley chloroplast mRNA stability.

Base Sequence↗
PubMed1992

A novel light-regulated promoter is conserved in cereal and dicot chloroplasts.

The chloroplast psbD-psbC genes encode D2 and cp43, a reaction center protein and chlorophyll-binding antenna protein of photosystem II, respectively. We have previously shown that differential accumulation of light-induced psbD-psbC mRNAs in barley chloroplasts is due to transcription from a blue light-responsive promoter (LRP). It is hypothesized that the light-induced mRNAs help to maintain levels of the D2 polypeptide, which is photodamaged and degraded in illuminated plants. To determine if light-induced accumulation of psbD-psbC mRNAs was a conserved phenomenon in chloroplasts, the expression of psbD-psbC operons from five cereals (barley, wheat, rice, maize, and sorghum) and three dicot (tobacco, spinach, and pea) species was examined. Cereal and dicot psbD-psbC operons differ due to several DNA rearrangements that moved psbK-psbI proximal to psbD-psbC, allowing cotranscription of these genes and production of several unique transcripts in cereals. Despite differences in the structure and expression of the cereal and dicot psbD-psbC operons, the accumulation of light-induced psbD-psbC mRNAs was conserved in all species studied. An unusual feature of the light-induced mRNAs was the occurrence of 5' end microheterogeneity. The multiple 5' termini were mapped to several consecutive nucleotides (8 to 25 bp) within a highly conserved (61%) DNA region that represents the transcription initiation site for the mRNAs in barley and tobacco. The novel LRP differs in sequence from typical plastid promoters that have prokaryotic "-10" and "-35" elements and is centered 570 bp (cereals), 900 bp (tobacco, spinach), or 1100 bp (pea) upstream from the psbD translational start codon. We propose that physiological and gene regulatory demands of the chloroplast act as constraints that preserved the linkage of the LRP with psbD despite DNA inversions involving the psbD upstream region.

Base Sequence↗
PubMed1991

A mixed group II/group III twintron in the Euglena gracilis chloroplast ribosomal protein S3 gene: evidence for intron insertion during gene evolution.

The splicing of a 409 nucleotide intron from the Euglena gracilis chloroplast ribosomal protein S3 gene (rps3) was examined by cDNA cloning and sequencing, and northern hybridization. Based on the characterization of a partially spliced pre-mRNA, the intron was characterized as a 'mixed' twintron, composed of a 311 nucleotide group II intron internal to a 98 nucleotide group III intron. Twintron excision is via a 2-step sequential splicing pathway, with removal of the internal group II intron preceding excision of the external group III intron. Based on secondary structural analysis of the twintron, we propose that group III introns may represent highly degenerate versions of group II introns. The existence of twintrons is interpreted as evidence that group II introns were inserted during the evolution of Euglena chloroplast genes from a common ancestor with eubacteria, archaebacteria, cyanobacteria, and other chloroplasts.

Amino Acid Sequence↗
PubMed1991

Intercistronic group III introns in polycistronic ribosomal protein operons of chloroplasts.

A novel ribosomal protein operon in the Euglena gracilis chloroplast genome was characterized. It encodes the genes for ribosomal proteins S4 and S11 (rps4 and rps11). The coding region of the rps11 gene is interrupted by two introns of 107 and 100 bp. The introns belong to a distinct class known as group III introns. The major transcript from this operon was characterized as a fully spliced dicistronic rps4-rps11 mRNA by RNA blot analysis, primer extension sequencing, and cDNA cloning and sequencing. An additional 95 nucleotide (nt) group III intron was identified in the 123 nt rps4-rps11 intercistronic region. The identification of the intercistronic intron between the rps4 and rps11 genes was unexpected. Other RNA transcripts from regions of the genome that could potentially contain intercistronic introns were re-examined and two other intercistronic, group III introns were found. These are located in a large ribosomal protein operon between the genes for the ribosomal proteins L23 and L2, and between L14 and L5. There are at least 50 group III introns in the E. gracilis chloroplast genome. All but 6 are found in genes encoding protein components of the transcriptional and translational apparatus. The distribution of group III introns and the unusual location of intercistronic group III introns may reflect some aspect of gene expression, or provide some insight into the mechanism of their splicing.

Amino Acid Sequence↗
PubMed1990

Light-induced switch in barley psbD-psbC promoter utilization: a novel mechanism regulating chloroplast gene expression.

The synthesis of reaction center protein D2 and mRNAs which encode this protein are differentially maintained at high levels in mature barley chloroplasts. To understand the differential maintenance of psbD mRNA abundance, we have studied the transcription and the RNAs produced from the psbD-psbC operon in plastids of light and dark-grown barley seedlings. Ten psbD-psbC RNAs synthesized in dark-grown barley share four different 5'-ends, two of which arise by transcription initiation, and one of which is generated by 5'-processing of longer psbD-psbC transcripts. Illumination of dark-grown barley causes the decline of these ten transcripts, and the accumulation of two different psbD-psbC RNAs. Capping assays, in vitro transcription and RNA processing experiments and treatment of plants with tagetitoxin (a selective inhibitor of chloroplast transcription), indicate that the light-induced transcripts arise by transcription initiation. Run-on transcription and RNA quantitation experiments provide evidence that both light-induced transcription and RNA stability play roles in the accumulation of the light-induced RNAs. These data document a novel mechanism for regulating plastid gene expression involving a light-induced switch in psbD-psbC promoter utilization.

Base Sequence↗
PubMed1990

Complex RNA maturation pathway for a chloroplast ribosomal protein operon with an internal tRNA cistron.

We have studied the expression of a large chloroplast ribosomal protein operon from Euglena gracilis that resembles the Escherichia coli S10 and spc ribosomal protein operons. We present evidence that 11 ribosomal protein genes, a tRNA gene, and a new locus, orf214/orf302, are expressed as a single transcription unit. The primary transcript also contains at least 15 group II and group III introns. Gene-specific probes for each ribosomal protein gene, orf214/orf302, and for trnl hybridized to a common pre-mRNA of an estimated size of 8.3 kilobases. This is the RNA size predicted for a full-length transcript of the entire operon after splicing of all 15 introns. Polycistronic ribosomal protein mRNAs accumulated primarily as spliced hepta-, hexa-, penta-, tetra-, tri-, and dicistronic mRNAs, which were presumed to arise by stepwise processing of the 8.3-kilobase pre-mRNA. A novel finding was the cotranscription of the trnl gene as an internal cistron within the ribosomal protein operon. Several combined mRNA/tRNA molecules, such as the pentacistronic rpl5-rps8-rpl36-trnl-rps14, were characterized. The occurrence of the orf214/orf302 is a unique feature of the Euglena operon, distinguishing it from all chloroplast and prokaryotic ribosomal protein operons characterized to date. The orf214/orf302 are not similar to any known genes but are cotranscribed with the ribosomal protein loci and encode stable RNA species of 2.4, 1.8, and 1.4 kilobases.

Animals↗
PubMed1989

Euglena gracilis chloroplast ribosomal protein operon: a new chloroplast gene for ribosomal protein L5 and description of a novel organelle intron category designated group III.

We describe the structure (3840 bp) of a novel Euglena gracilis chloroplast ribosomal protein operon that encodes the five genes rpl16-rpl14-rpl5-rps8-rpl36. The gene organization resembles the spc and the 3'-end of the S10 ribosomal protein operons of E. coli. The rpl5 is a new chloroplast gene not previously reported for any chloroplast genome to date and also not described as a nuclear-encoded, chloroplast protein gene. The operon contains at least 7 introns. We present evidence from primer extension analysis of chloroplast RNA for the correct in vivo splicing of five of the introns. Two of the introns within the rps8 gene flank an 8 bp exon, the smallest exon yet characterized in a chloroplast gene. Three introns resemble the classical group II introns of organelle genomes. The remaining 4 introns appear to be unique to the Euglena chloroplast DNA. They are uniform in size (95-109 nt), share common features with each other and are distinct from both group I and group II introns. We designate this new intron category as 'group III'.

Amino Acid Sequence↗
PubMed1988

Organization of the psbE, psbF, orf38, and orf42 gene loci on the Euglena gracilis chloroplast genome.

The genes for cytochrome b559, designated psbE and psbF, and two highly conserved open reading frames of 38 and 42 codons have been located and characterized on the chloroplast genome of Euglena gracilis. The organization of the genes is psbE - 8 bp spacer - psbF - 110 bp spacer - orf38 - 87 bp spacer - orf42. All genes are of the same polarity. The psbE gene contains two introns of 350 and 326 bp. The psbF gene contains a single large intron of 1,042 bp. The orf38 and orf42 loci lack introns. The introns are extremely AT rich with a pronounced base composition bias of T greater than A greater than G greater than C in the mRNA-like strand and group II-like boundary sequences at their 3' and 5' ends having the consensus 5'-GTGTG .. INTRON .. TTAATTTNAT-3'. The psbE gene consists of 82 codons and encodes a polypeptide with a predicted molecular weight of 9,212. The psbF gene consists of 42 codons, which specify a polypeptide with a predicted molecular weight of 4,785. The highly conserved open reading frames of 38 and 42 codons code for polypeptides with predicted molecular weights of 4,405 and 4,426, respectively. The gene products of psbE, psbF, orf38 and orf42 are, respectively, 69.5%, 70% and 61.5% identical to those found in higher plants. The predicted secondary structure of the proteins from hydropathy plots is consistent with each containing a single membrane-spanning domain of at least 20 amino acids. Each of the genes is preceded by sequences which may serve as ribosome binding sites. All four genes are transcribed.

Amino Acid Sequence↗
PubMed1988

Organization of ribosomal protein genes rpl23, rpl2, rps19, rpl22 and rps3 on the Euglena gracilis chloroplast genome.

The nucleotide sequence (4,814 bp) was determined for a cluster of five ribosomal protein genes and their DNA flanking regions from the chloroplast genome of Euglena gracilis. The genes are organized as rpl23-150 bp spacer-rpl2-59 bp spacer-rps19-110 bp spacer-rpl22-630 bp spacer-rps3. The genes are all of the same polarity and reside 148 bp downstream from an operon for two genes of photosystem I and four genes of photosystem II. The Euglena ribosomal protein gene cluster resembles the S-10 ribosomal protein operon of Escherichia coli in gene organization and follows the exact linear order of the analogous genes in the tobacco and liverwort chloroplast genomes. The number and positions of introns in the Euglena ribosomal protein loci are different from their higher plant counterparts. The Euglena rpl23, rps19 and rps3 loci are unique in that they contain three, two and two introns, respectively, whereas rpl2 and rpl22 lack introns. The introns found in rpl23 (106, 99 and 103 bp), rps19 (103 and 97 bp) and rps3 intron 2 (102 bp) appear to represent either a new class of chloroplast intron found only in constitutively expressed genes, or possibly a degenerate version of Euglena chloroplast group II introns. They are deficient in bases C and G and extremely rich in base T, with a base composition of 53-76% T, 25-34% A, 3-10% G and 2-7% C in the mRNA-like strand. These six introns show minimal resemblance to group II chloroplast introns. They have a degenerate version of the group II intron conserved boundary sequences at their 5' and 3' ends. No conserved internal secondary structures are apparent. By contrast, rps3 intron 1 (409 bp) has a potential group II core secondary structure. The five genes, rpl23 (101 codons), rpl2 (278 codons), rps19 (95 codons), rpl22 (114 codons) and rps3 (220 codons) encode lysine-rich polypeptides with predicted molecular weights of 12,152, 31,029, 10,880, 12,819, and 25,238, respectively. The Euglena gene products are 18-50%, and 29-58% identical in primary structure to their E. coli and higher plant counterparts, respectively. Oligonucleotide sequences corresponding to Euglena chloroplast ribosome binding sites are not apparent in the intergenic regions. Inverted repeat sequences are found in the upstream flanking region of rpl23 and downstream from rps3.

Amino Acid Sequence↗