Biomedical subjects

L K Read

Publications and source records attributed to L K Read.

24 records · Page 2Linked to original sources

PubMed1992

Chimeric and truncated RNAs in Trypanosoma brucei suggest transesterifications at non-consecutive sites during RNA editing.

RNA editing adds and removes uridines at specific sites in several mitochondrial transcripts in kinetoplastid parasites probably as specified by guide RNAs (gRNAs) that are complementary to the final edited sequence. Editing has been postulated to involve transesterification which predicts (1) chimeric molecules with a gRNA covalently attached by its non-encoded oligo U tail to an internal editing site in the mRNA and (2) the corresponding truncated 5' portions of the mRNAs. We have characterized cDNAs representing a large number of both types of intermediates from Trypanosoma brucei. The lengths of both U tails and encoded gRNA sequences vary greatly in length. The majority of encoded gRNA sequences are shorter than predicted based on their minicircle coding sequences. Analysis of the predominant sites of gRNA attachment in chimeras suggests that the transesterifications that religate the truncated 5' mRNAs may proceed more rapidly at editing sites at the 5' end of an editing domain and at sites of U deletion. Partially edited sequences in the mRNA portion of chimeras and at the 3' ends of truncated 5' mRNAs also indicate a non-consecutive order of site selection during RNA editing.

Animals↗

PubMed1992

Extensive editing of both processed and preprocessed maxicircle CR6 transcripts in Trypanosoma brucei.

Transcripts from several genes encoded in the Trypanosoma brucei maxicircle genome are altered by posttranscriptional uridine insertion and deletion through a process called RNA editing. We find that transcripts from the CR6 gene are extensively edited by addition of 132 uridines and deletion of 28 uridines to produce a fully edited mRNA 47% larger than unedited mRNA. Two open reading frames (ORFs) and their initiation and termination codons are created by editing of CR6 mRNA. Both ORFs specify small, hydrophobic proteins with no homology to proteins in three databases. Both unedited and edited CR6 transcripts are more abundant in bloodstream form than in procyclic form parasites. cDNA clones spanning both CR6 and the downstream NADH dehydrogenase subunit 5 (ND5) gene were isolated, indicating that mature CR6 and ND5 transcripts arise from a common precursor. Sequencing of these cDNAs revealed 37 nucleotides of overlap between the 3' end of CR6 and the 5' end of ND5. In addition, the CR6 portion of many of these molecules was extensively edited, indicating that RNA editing can precede precursor processing. These results provide the first clear demonstration of polycistronic transcription of maxicircle genes, and suggest new mechanisms by which both RNA editing and precursor processing may regulate maxicircle gene expression.

Amino Acid Sequence↗

PubMed1991

Cycles of progressive realignment of gRNA with mRNA in RNA editing.

We characterized numerous partially edited NADH dehydrogenase 7 and ATPase 6 cDNAs. Most of these have a stretch of incompletely edited sequence at the junction of mature and unedited sequences. The characteristics of the junctions suggest editing of sites multiple times and that editing within each junction does not proceed precisely 3' to 5'. Analyses of gRNAs and corresponding junction sequences predict a series of progressively more stable, but incompletely base-paired, interactions in the junction region. The predicted interactions suggest that the gRNA is progressively realigned with the mRNA being edited. We suggest that gRNA interactions with the mRNA result in regions of lower thermodynamic stability that are selected for editing, thus driving toward the most stable structure, the complete gRNA/mRNA duplex.

Adenosine Triphosphatases↗

PubMed1991

Plasmodium falciparum-infected erythrocytes contain an adenylate cyclase with properties which differ from those of the host enzyme.

It has been postulated that differentiation of the human malaria parasite, Plasmodium falciparum, is controlled by cAMP levels. We have determined that P. falciparum synthesizes an adenylate cyclase with several properties distinct from those of the mammalian host cell enzyme. Adenylate cyclase activity was compared in P. falciparum-infected erythrocytes, isolated parasites free of host cell material, and uninfected erythrocyte membranes. The parasite enzyme was unaffected by GTP gamma S, AlF4-, and forskolin, while the erythrocyte enzyme was markedly stimulated by each of these compounds. The parasite adenylate cyclase also exhibited a striking preference for Mn2+ over Mg2+, which was not evident in the erythrocyte enzyme. Moreover, differing cation and pH sensitivities were observed for adenylate cyclase activity in the two cell types. When infected and uninfected erythrocytes were compared, the basal adenylate cyclase activity of infected cells was 7 and 49 times that measured in uninfected erythrocytes in the presence of Mg2+ and Mn2+, respectively. Furthermore, adenylate cyclase activity in infected cells exhibited properties typical of the parasite enzyme. This indicates that synthesis of the parasite enzyme rather than stimulation of the host enzyme accounts for the increased activity in infected cells.

Adenylyl Cyclases↗

PubMed1991

Comparison of adenylate cyclase and cAMP-dependent protein kinase in gametocytogenic and nongametocytogenic clones of Plasmodium falciparum.

Adenylate cyclase and cAMP-dependent protein kinase activities in gametocytogenic (LE5) and nongametocytogenic (T9/96) clones of Plasmodium falciparum were compared to explore the role of cAMP in sexual differentiation of the parasite. Basal adenylate cyclase levels were equivalent in the 2 clones. However, cAMP-dependent histone II-A kinase activity was significantly higher in LE5 than in T9/96 over a range of cAMP concentrations. This difference was due to a decreased Vmax for the enzyme in the nongametocytogenic clone and not to an increased Ka for cAMP. Examination of parasite cAMP-binding proteins, likely to be kinase regulatory subunits, by both photoaffinity labeling with [32P]8-N3-cAMP and affinity chromatography of metabolically [35S]methionine-labeled cytosol of cAMP-agarose revealed a 53-kDa cAMP binding protein in both clones and a 49-kDa cAMP-binding protein in T9/96 that was absent in LE5. Our results suggest that T9/96 has lost the ability to undergo gametocytogenesis due to a substantial decrease in cAMP-dependent protein kinase activity rendering the parasite unable to respond to increased intracellular cAMP levels. Moreover, the reduction in cAMP-dependent protein kinase activity may be due to the presence of an alternative regulatory subunit of the kinase.

Adenylyl Cyclases↗

PubMed1990

Cyclic AMP- and Ca2(+)-dependent protein kinases in Plasmodium falciparum.

The cyclic AMP- and Ca2(+)-dependent protein kinase activities of Plasmodium falciparum were partially characterized after purification of parasites from host erythrocytes by N2 cavitation and Percoll gradient centrifugation. Proteins of molecular weights 80, 54, 51, and 31.5 kDa were phosphorylated in a cAMP-dependent manner in cytosolic extracts of isolated P. falciparum. Cytosolic extracts also contained cAMP-dependent histone II-A kinase activity with an average Vmax of 131.1 pmol/32P/min/mg protein and a Km for cAMP of 85nM. Upon photoaffinity labeling with [32P]-8-N3-cAMP, a 53-kDa protein was specifically labeled in parasite cytosol. A metabolically labeled protein of the same molecular weight was identified by cAMP-agarose affinity chromatography. The 53-kDa protein cochromatographed with cAMP-dependent histone II-A kinase activity on DEAE-cellulose, suggesting that it is the regulatory subunit of the kinase. Ca2(+)-dependent phosphorylation of proteins of molecular weights 195, 158, 51, 47.5, and 15 kDa was demonstrated in a membrane fraction from parasites free of the erythrocyte membrane. This activity was not stimulated by either calmodulin or phospholipid plus diacylglycerol and was absent from the membranes of uninfected erythrocytes. Of several exogenous substrates tested, none were found to be a substrate for this Ca2(+)-dependent kinase. Both cAMP- and Ca2(+)-dependent kinases phosphorylated serine and threonine residues.

Animals↗