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Biomedical subjects

P M Lizardi

Publications and source records attributed to P M Lizardi.

44 records · Page 3Linked to original sources
PubMed1979

Discontinuous translation of silk fibroin in a reticulocyte cell-free system and in intact silk gland cells.

Silk fibroin mRNA was translated in a rabbit reticulocyte cell-free system. Addition of tRNA from silk glands was essential for complete translation of the fibroin polypeptide. (Mr approximately 400,000). Synthesis of full-sized product took at least 85 min. In addition to full-size product, a large number of smaller polypeptides were observed upon analysis by sodium dodecyl sulfate/polyacrylamide gel electrophoresis. Evidence is presented that these smaller polypeptides are growing fibroin chains that transiently accumulate as discrete size classes due to discontinuities in the translation process. These discontinuities, or pauses, occur at specific sites in the fibroin mRNA template. The relative duration of the pauses can be experimentally modulated by changing the source of the supplementary tRNA added to the in vitro system. Silk glands were incubated in organ culture under conditions where essentially exclusive labeling of newly synthesized fibroins was attained. Analysis in sodium dodecyl sulfate gels showed that the labeling pattern of nascent silk fibroins is similar to the pattern observed in the reticulocyte cell-free system. This result suggests that discontinuities or pauses in polypeptide chain elongation also occur in vivo under conditions of organ culture.

Animals↗
PubMed1976

The size of pulse-labeled fibroin messenger RNA.

A method has been developed for the isolation of fibroin gene transcripts from total RNA of the silkworm Bombyx mori. It is based on affinity chromatography using Sephadex-bound polynucleotides capable of selectively hybridizing with fibroin mRNA sequences. In vivo pulse labeling of the posterior silk gland for periods of 10-35 min produces labeled heterogeneous nuclear RNA of high molecular weight (greater 40S). Fibroin gene transcripts can be selected from the total hnRNA population by two consecutive passages through the affinity column. Analysis of the column-bound material in denaturing polyacrylamide-agarose gels reveals that the size of pulse-labeled fibroin mRNA is essentially the same (within 5%) as that of mature cytoplasmic mRNA. This holds true for pulses as short as 6 min, where even nascent mRNA can be observed. However, a small shoulder of material is present on the heavy side of the pulse-labeled mRNA, which could be indicative of an extremely short-lived precurosr species. The purified pulse-labeled mRNA (10 min incorporation) has been further analyzed by chromatography in oligo(dT)-cellulose. The data show that the mRNA is polyadenylated within a few minutes after synthesis.

Animals↗
PubMed1975

The size of fibroin messenger RNA and its polyadenylic acid content.

The molecular weight of fibroin messenger RNA has been measured by its contour length in the electron microscope and its electrophoretic mobility under conditions of complete denaturation. These methods give values of 5.6 times 10-6 and 6.0 times 10-6 daltons, respectively. The average molecular weight of 5.8 times 10-6 (1.6 times 10-4 base residues) would be expected to code for a protein of 414,000 daltons, a value about 12 percent larger than the most recently published size for fibroin. When the mRNA is prepared carefully to avoid shear breakage, it is as homogeneous in size as ribosomal RNA. Between 43 percent and 81 percent of the mRNA molecules bind to oligo(dT)-cellulose and are presumed to contain a poly(A) sequence. This sequence, isolated after ribonuclease digestion, comprises about 0.6 percent of the molecule, or an estimated 100 nucleotides. The poly(A) has been localized at the end of the molecule by hybridizing with a small poly (dT:dA) piece and subsequent visualization by electron microscopy. This observation, along with the ability of reverse transcriptase to transcribe fibroin mRNA with an oligo(dT) primer, demonstrates that the poly(A) is at the 3' end of the mRNA.

Adenine Nucleotides↗
PubMed1975

The length of the fibroin gene in the Bombyx mori genome.

Radioactive anti-messenger DNA (3H-cDNA) complementary to silk fibroin mRNA has been synthesized using reverse transcriptase. This 3H-cDNA has been found to be a specific and sensitive probe for the detection of fibroin genes in the genome of Brombyx mori. Actinomycin-CsCl gradients give a large separation of the high GC fibroin genes from the bulk DNA. This density shift of fibroin genes has been measured as a function of DNA molecular weight. The data support a model in which a single high GC fibroin gene of 11.6 times 10-6 daltons is surrounded by at least 6 times 10-7 daltons of low GC DNA (30-39 percent). This finding, along with saturation hybridization studies (Suzuki, Gage, and Brown, 1972), demonstrate that the fibroin gene is present in a single copy per haploid genome.

Animals↗
PubMed1972

The intracellular site of synthesis of mitochndrial ribosomal proteins in Neurospora crassa.

The intracellular site of synthesis of mitochondrial ribosomal proteins (MRP) in Neurospora crassa has been investigated using three complementary approaches. (a) Mitochondrial protein synthesis in vitro: Tritium-labeled proteins made by isolated mitochondria were compared to (14)C-labeled marker MRP by cofractionation in a two-step procedure involving isoelectric focusing and polyacrylamide gel electrophoresis. Examination of the electrophoretic profiles showed that essentially none of the peaks of in vitro product corresponded exactly to any of the MRP marker peaks. (b) Sensitivity of in vivo MRP synthesis to chloramphenicol: Cells were labeled with leucine-(3)H in the presence of chloramphenicol, mitochondrial ribosomal subunits were subsequently isolated, and their proteins fractionated by isoelectric focusing followed by gel electrophoresis. The labeling of every single MRP was found to be insensitive to chloramphenicol, a selective inhibitor of mitochondrial protein synthesis. (c) Sensitivity of in vivo MRP synthesis to anisomycin: We have found this antibiotic to be a good selective inhibitor of cytoplasmic protein synthesis in Neurospora. In the presence of anisomycin the labeling of virtually all MRP is inhibited to the same extent as the labeling of cytoplasmic ribosomal proteins. On the basis of these three types of studies we conclude that most if not all 53 structural proteins of mitochondrial ribosomal subunits in Neurospora are synthesized by cytoplasmic ribosomes.

Carbon Isotopes↗