Date of Award

1986

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Abstract

Extracts of sporangiospores of Mucor racemosus contained RNA that readily hybridized with ('3)H polyuridylic acid. Prior to germination, this RNA was in a form sedimenting at <80S. Within 10 minutes after initiating germination, most of this RNA sedimented with polyribosomes and 80S monoribosomes. Particulate material from spore extracts bound to oligo dT -cellulose at high ionic strength and was assumed to contain messenger ribonucleoprotein particles (mRNP's). A portion of the mRNP's was released from the column by lowering the ionic strength. Other portions were eluted stepwise in buffer containing 50% and 90% formamide and in 0.1-N NaOH. Identical elution patterns were observed whether monitoring incorporated ('31)P-orthophosphate or L- ('32)S methionine, absorbance at 280 nm, or hybridization of ('3)H polyuridylic acid. mRNP's from the first two fractions were analyzed. A bimodal population of particles was detected in sedimentation velocity and sedimentation equilibrium centrifugation. Particles eluted at low ionic strength demonstrated a sedimentation coefficient distribution of 20S-to-80S, with a mean of 55S. Particles eluted in formamide demonstrated a sedimentation coefficient distribution of 20S-to-60S, with a mean of 40S. Particles eluted at low ionic strength displayed two peaks in CsCl centrifugation, with bouyant densities of 1.37 gm/cc and 1.59 gm/cc. Particles eluted in formamide displayed a single peak with a buoyant density of 1.61 gm/cc. Particles eluted at low ionic strength and centrifuged in metrizamide solution formed two bands having bouyant densities of 1.15 gm/cc and 1.30 gm/cc; formamide-eluted particles banded only at the higher density. Mucor 40S ribosomal subunits banded at 1.56 gm/cc and 1.28 gm/cc in CsCl and metrizamide solution respectively. Analysis of mRNP fractions by sodium dodecylsulfate-polyacrylamide gel electrophoresis characterized protein components of these particles which helped explain their biomodal sedimentation behaviour. Each mRNP fraction contained a unique spectrum of proteins not shared by ribosomes or soluble cell proteins. The formamide-eluted particles contained two proteins, whereas the particles eluted at low ionic strength possessed 12 proteins. A 24,000-dalton protein was the predominant form in both. The denser mRNP's were smaller and lacked proteins found in the larger less dense particles. The former had a protein/RNA ratio of 45/55; whereas the later possessed a value of 90/10. Messenger RNA extracted from the mRNP's sedimented between 8S-to-20S.

Pages

172

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