Download the efficient expression of a eukaryotic gene in a prokaryotic cell free

WEDNESDAY 1 APRIL
POSTERS RELATED TO S17
Wed-S17-10
Wed-S17-11
THE EFFICIENT EXPRESSION OF A EUKARYOTIC GENE IN
A PROKARYMIC CELL FREE SYSTEM
James F. Glover and T. Michael A. Wilson
CONTROL OF PROTEIN SYNTHESIS I N FPV INFECTED CELLS
0. Evans and C . S c h o l t i s s e k
I n s t i t u t f u r Virologie.
Department of Biochemistry, University of
Liverpool, P.0.BOx 147, Liverpool L69 3BX, U.K.
Translation of genomic and 3'-teminal subgenomic
TMV RNA fragments in a cell free system derived
from E.coli (MRE 600) gives rise to polypeptides
in the 10-50 Kd range of molecular weight as
determined on SDS-PAGE. Two major polypeptides of
30K and 17.5K correspond to products from cistrons
mapped internally on polycistronic TMV RNA. The
more pronounced product at 17.5K comigrates with
authentic TMV coat protein. Peptide mapping techniques confirm it to be T V coat protein with an
extra N-terminal fmet residue as shown by
differential labelling patterns. This product can
be assembled into nucleoprotein particles. The
structure of the 70s ribosome binding site at a
normally silent, internal cistron on 27s TMV RNA
has been investigated. The valve of TMV RNA as a
model for eukaryotic gene expression in
prokaryotic systems is discussed.
F r a n k f u r t e r S t r a s s e 107,
6300 G i e s s e o , W e s t Germany
P r o t e i n synthesis by a
3 mutant
o f fowl plague v i r u s ,
t s 236, which contains a mutation i n t h e gene coding f o r
t h e P3 p o l y p e p t i d e , was i n v e s t i g a t e d . Synthesis o f t h e
HA polypeptide could n o t be detected i n chick embryo
f i b r o b l a s t s i n f e c t e d a t the r e s t r i c t i v e temperature. The
HA could be d e t e c t e d i m n e d i a t e l y on s h i f t down t o t h e
permissive temperature even when f u r t h e r mRNA synthesis was
prevented by Actinomycin 0. The synthesis o f f u n c t i o n a l
HA mRNA a t t h e r e s t r i c t i v e temperature was demonstrated
by i n v i t r o t r a n s l a t i o n and h y b r i d i z a t i o n . Thus t h e P3
polypeptide appears t o have a r o l e i n t h e c o n t r o l o f t r a n s l a t i o n o f HA i n FPY i n f e c t e d c e l l s .
Wed-S 17-12
Wed-S17-13
RELATIVE RATES OF DEGRADATION OF SERINE tRNA
SPECIES DURING VITELLOGENIN INDUCTION IN AVIAN
LIVER. Pirkko A. Kanerva, Dept. of Biochemistry,
Univ. of Kuopio, SF-70101 Kuopio 10, Finland
EVIDENCE FOR LOW MOLECULAR WEIGHT RNA AS CONTROL
ELEMENT OF THE TRANSLATIONAL EFFICIENCY OF RNA
FROM RAT LIVER NON-POLYSOMAL mRNP PARTICLES
Barbara Kiihn, Wolfgang Northemann, and Peter C.
Heinrich, Biochemisches Institut der Universitat
Freiburg, 7800 Freiburg, Germany
The hepatic synthesis of vitellogenin, a serinerich yolk protein precursor, induced by estrogens
is accompanied by an increase in the serine acceptance of unfractionated liver tRNA. No specific
increase in the relative rates of synthesis of the
two major Ser-tRNA species occurs during vitellogenin induction. The present experiments were performed to assess the relative rates of degradation
of the two Ser-tRNA species in estrogen-treated
and control roosters. The method was an in vivo
pulse-chase labeling with radioactive orotate and
purification of the Ser-tRNA species by chromatography. The results indicate that the rate of degradation of Ser-tRNA (AGU,AGC), the isoacceptor
which is preferentially attached to membrane-bound
ribosomes, is slowed down relative to Ser-tRNA
(UCU,UCC,UCA). A role for specific degradation in
adaptation of the Ser-tRNA species is suggested.
When phenol-extracted RNA from 205 or 40s mRNP
particles is translated in an in vitro system
from wheat germs, a strong inhibition of 13Hlleucine incorporation into proteins i s observed at
high RNA concentrations. This inhibitory effect
was not observed, when the low molecular weight
RNA (ImwRNA) of < 8s had been separated, indicating that lmwRNA may be responsible for the inhibition. A similar, linear translation behaviour
was found, when the phenol-extracted RNA from
405 mRNP particles 6 h after inhibition of transcription was used.
It is proposed that lmwRNA with a rather high
turnover, present in very low concentrations,
affects the translatability of mRNA from 40s
mRNP particles.
Wed-S17- 14
Wed-S17-15
DETERMINATION OF FREE AMINO ACIDS AND AMINO ACIDS
ATTACHED TO tRNA IN TISSUES BY GAS-LIQUID CHROMATOGRAPHY. Leena K. Lindqvist and P.H. Mgenpaa,
Dept. of Biochemistry, Univ. of Kuopio, SF-70101
Kuopio 10, Finland
PRESENCE OF A PROTEIN SYNTHESIS- INHIBITING
FACTOR OF BOTH STAGES OF DEVELOPMENT OF THE
SILKMOTH ANTHEREA PERNYI
LGZFrago_y&iz and P.Traub
Dept.of Biol. Athens Univ. Kouponia 621 Greece
M.P.I. fur Zellbiologie 6802 Ladenburg w.Germany
Cell-free protein synthesizing systems from insects
are a useful tool for translational control studies.
Analyzing the S
fractions prepared from oocytes
of two differengostages of development we found a
factor (specific RNAse?) which completely inhibits
the translation of Hb mRNA, and partially the
translation of poly(U), in these protein synthesizing
either to proteinsynthesystems. Addition of S
sizing polysomes or to3fhe wheat germ system
results in an 85% decrease of the protein synthetic
activity. Radioactive mRNA as well as28S and 18s
rRNA are not degraded tg oligonucleotides during
40 min incubation at 25 C in Sgo.However,sucrose
gradient centrifugation of these mixtures revealed
that the mRNA as well as the 18s and 28s rRNA
sediment with lower S values as well defined peaks.
Methods were developed to quantitatively analyze free amino acids and amino acids attached to
tRNA in tissue samples by gas-liquid chromatography. Free amino acids were purified by ion-exchange chromatography after deproteinization.Tota1
cellular aminoacyl-tRNA was extracted by a modified phenol extraction method under conditions
which were designed to prevent deacylation of the
attached amino acids. After deacylation and purification, the amino acids were determined by gasliquid chromatography as their N-heptafluorobutyryl n-propyl derivatives using packed and glasscapillary columns. Distinct differences were observed between aminoacyl-tRNAs derived from rabbit reticulocytes and liver.
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