close

Вход

Забыли?

вход по аккаунту

?

Determination of the Sequence of a Code Triplet.

код для вставкиСкачать
The Photochemical Decomposition of Alkyl
Azidoformates in Aliphatic Alcohols
Determination of the Sequence of a Code Triplet
By Prof. Dr. F. Cramer, Dr. H. Kuntzel, and
Dr. J . H. Matthaei [*]
By Dr. R. Kreher and Dip1.-lng. G. H. Bockhorn
lnstitut fur Organische Chemie der Technischen Hochschule
Darmstadt (Germany)
Medizinische Forschungsanstalt
der Max-Planck-Gesellschaft, Gottingen (Germany)
Dedicutrd to Professor C . Sclzopf on the occasioiz of his 65th
hirthduy
Up to the present, only polyribonucleotides with a statistical
distribution of their bases were available for deciphering the
genetic code; hence only the overall composition of most of
the code triplets could be determined [1,2]. Recently, we
have indicated possibilities for further analysis of the genetic
code using oligonucleotides and polynucleotides with definite
base sequences 131.
An enzymatic method was used t o obtain coding polynucleotides with definite 3'-terminal base sequences. As expected [4], these polynucleotides effect specific adsorption of
aminoacyl-RNA onto ribosomes in a simplified cell-free
system derived from Escherichiu coli [4]. This enabled us to
carry out the first determination of the base sequence of a
coding triplet. This finding also demonstrates that the R N A
template is read off starting from the 3'-terminal end.
The following polynucleotides were prepared using a partially purified polynucleotide phosphorylase from MicroCOCCUS [ysodrikticus [ 5 ] : poly-pApC 50: 1 ( I ) , poly-pApU
50: I (2), poly-pApG 100: I ( 3 ) , poly-pUpG 100: 1 ( 4 ) , and
poly-pCpG IO0:l (5). Polynucleotides ( I ) and (2) were
hydrolysed with pyrimidine-specific ribonuclease from pancreas, and (3), (41, and (5) with guanine-specific ribonuclease
T1 from takadiastase. The 3'-phosphate residues were removed from the cleavage products using bacterial alkaline
phosphatase.
For the polynucleotide Ap...ApApC obtained from ( I ) the
3'-terminal nucleotide was determined as follows (this polynucleotide contains a n average of 50 nucleotides per chain):
Samples of the polynucleotide were labelled in the cytosine
moiety with tritium, and the [3H]cytosine was liberated by
oxidation with periodate and subsequent treatment with
cyclohexylamine [6]. Equilibrium dialysis for 6 h against
water showed that all the radioactivity had been liberated
in the form of dialysable nucleobase, while the remainder
of the ultraviolet-absorbing material retained by the dialysis
membrane. Examination of the product obtained from the
treatment with periodate and amine by paper chromatography revealed cytosine and no adenine.
About 150p.ynioles of ribosomes [8] were incubated for 60 sec
at 36 "C with 300 ppmoles of polynucleotide and 1 mpmole
of aminoacyl-RNA in 350 pl of standard buffer [7]. The
aminoacyl-RNA was charged with one labelled and 19 nonlabelled amino acids; all the 20 amino acids normally occurring in proteins were tested. Each incubate was then
diluted with 30 times its volume of ice-cold 0.01 M tris-HCI
buffer (pH 7.2)/0.01 M magnesium acetate and centrifuged
at 150000 g for 60 min. The precipitate obtained was taken
up in 0.1 M triethylammonium hydrogen carbonate (pH 7.6),
applied to fibre glass paper (Whatman GFA), and dried for
liquid scintillation counting. Comparison of the coding
properties of poly-Up..,UpUpG and poly-Up...UpU indicates that UpUpG is the coding sequence for leucine. However,
valine and cysteine, having also codons consisting of one
guanylic and two uridylic acid residues [2], are not coded by
this sequence (see Table; the values given are the mean
from two experiments).
Ethyl azidoformate (1) loses nitrogen during its photochemical decomposition [ I ] in t-butanol, and reacts with the tertiary alcohol to yield ethyl N-t-butoxycarbamide (2d), b.p.
97-98 'C/13 mni, in 60 % yield; (2d) is also obtained from
t-butoxyamine and ethyl chloroformate in the presence of
triethylamine. On photollsis in methanol, ethanol, or isopropanol, ( I ) is chiefly reduced to ethylcarbamide (3),
with simultaneous loss of nitrogen; according to gas chro- ( 2 c ) are also
matography, ethyl N-alkoxycarbamates (2~1)
formed in small quantities.
(2a), R = CH3-
(3J, 52% in methanol
28%
1-76), R = CHs-CHz( Z C ) , R = (CH3)ZCH(2d), R = (CH,),C-
97% in ethanol
3%
10%
60%
.G
90% in isopropanol
y
1
-9C = O
The photochemical decomposition of ( I ) presumably proceeds by way of the reactive ethoxycarbonylazene ( I u) [2]. In
methanol, ethanol, and isopropanol, ( I u) apparently dehydrogenates the solvent to give the corresponding carbonyl
compound, being thereby reduced to ( 3 ) [3].
O n photolysis of the t-butyl ester (4) in t-butanol, the tbutoxycarbonylazene ( 4 a ) initially formed reacts not only
with the tertiary alcohol to yield t-butyl N-t-butoxycarbamide
( 4 d ) , m.p. 70-71 " C ,in 29 % yield, but preferentially tends to
undergo a n intramolecular reaction to yield 6 0 % 5,5dimethyloxazolid-2-one (5), m.p. 80-82OC [4]; in this reaction, the azene group is apparently-inserted into a C-H bond
of the t-butyl group.
Received, July 3rd, 1964
[ Z 7701591 IE]
German version: Angew. Chem. 76, 681 (1964)
~~
[ I ] Refluxing alcoholic solutions of ( I ) were illuminated by
means of a high-pressure ultraviolet lamp [Type S 81, Quarzlampengesellschaft Hanau (Germany)]. Comparable results were
obtained by using a low-pressure ultraviolet lamp (Type N K
6/20, same source) at room temperature.
[2] K . Hafner and C . Konig, Angew. Chem. 75, 89 (1963); Angew. Chem. internat. Edit. 2, 96 (1963); U'. Lwowski and T. W .
Muttingly, Tetrahedron Letters 1962, 211; G. Jager, Ph. D.
Thesis, Technische Hochschule Darmstadt, 1962.
[31 Compare the photochemically induced reduction of benzophenone to benzhydrol by isopropanol: A. Sch~n~erg;Praparative
Organische Photochemie. Springer, Heidelberg 1958, p. 115.
[41 Photolysis of ( 4 ) in acetonitrile affords (5) in yields of up
to 80 ";b.
Angew. Chem. internat. Edit.
Vol. 3 (1964) I No. 8
Amino
acid
[14ClVal
['5S]Cys
['H]I.eu
PHIPhe
I without
139
482
780
64
Aminoacyl-RNA bound [nwnoles]
1
125
465
788
174
Z:hy-Up...UpUpG
350
480
716
70
Hydrolysis at p H 9 of the complex of ribosomes, polynucleotide, and aminoacyl-RNA obtained by centrifugation, and
subsequent high-voltage paper electrophoresis in 2 M acetic
589
acid showed tha t n o labelled am in o acid is linked by a
peptide bond in th e complex. Electron-microscopic investigations [9] of fractionated polyuridylic acid (average
chain length 750 A) indicated that 30-S- an d 50-S-subunits
of the ribosomes ad d c n t o one end of this nucleic acid
more tha n 12 times as much as o n to the rest o f the chain.
T he above observations suggest that this end is the 3’-terminal
one.
Received, July 31st, 1964
[Z 792/601 l E ]
German version: Angew. Chein. 76, 716 (1964)
~
_
_
[*I We are indebted to Misses E. Garttiw, K. Eckerr, and G.Heller
for valuable assistance.
[I] P. Lengyel, J. F. Speyer, and S . Ochoo, Proc. nat. Acad. Sci.
USA 47, 1936 (1961).
(21 J . H. Mntthaei, 0. W. Jones, R . T . Martin, and M . W. Nirenberg, Proc. nat. Acad. Sci. USA 48, 666 (1962).
[3] J. H. Matthaei, Nova Acta Leopoldina N. F. 26, 45 (1963).
[4] 1. H . Matthad, L.ecture at the Bunsen-Tagung, Berlin, May
1964.
[5] M. F. Singer and J. K. Cuss, J. biol. Chemistry 237, 182 ( I 962).
[6] P. A. Whitfeld, Biochem. .I. 58, 39 (1962).
[7] J. H . Matthaei and M . W . Nirenberg. Proc. nat. Acad. Sci.
USA 47, 1580 (1961).
[8] To avoid losses of the 3’-terminal nucleotide during incubation, the ribosomes must be purified from nucleolytic activity a s far a s possible.
[9] F. Arnelunxen and J. H . Matthaei, Lecture at the VIth International Congress of Biochemistry, New York, July 1964.
Coding with Mono- and Polytrinucleotides
By Dr. J. H. Matthaei, Dr. H. Kleinkauf, and
Prof. Dr. G . Schramm [*I
Medizinische Forschungsanstalt der Max-Planck-Gesellschaft, Gottingen, Botanisches Institut der Technischen
Hochschule Braunschweig, a n d Max-Planck-lnstitut fur
Virusforschung, Tiibingen (Germany)
Polytrinucleotides ought t o act as templates in th e biosynthesis
of poly(amino acids) [I], whereas isolated trinucleotides lead
only t o a more or less specific binding of aminoacyl-RNAs
onto ribosomes [I-31. Trinucleotides which would be inactive in a homopolymer because of its tertiary structure
might b e active in a copolymer with a second trinucleotide
[I]. W e have therefore started to prepare homopolytrinucleotides by chemical synthesis a n d to test their coding
properties. W e have found that A p G p C an d A p G p U ar e
coding sequences for asparagine.
A mixture of trinucleotides was secured by complete degradation of a commercial sample of yeast R N A with pancreatic
ribonuclease (RNase) followed by five extractions with
phenol, threefold extraction with ether, an d lyophilization.
Individual mono-, di-, an d trinucleotides were separated f r o m
6.5 g of t h e crude mixture of degradation products by
chromatography o n four columns (6 c m in diameter, 2 in
long) of DEAE-Sephadex (A-25 coarse) connected in series.
Movable pistons fitted with a sintered-glass filter were inserted into t h e columns from beneath in order t o keep the
Sephadex together even o n shrinkage. Th e elution was carried
o u t upwards (against gravity) using 4 x 2 channels of a
D urrum elastic-tubing p u m p , which pumped 165 I of a mmo nium carbonate solution (pH 8.7, concentration increasing
linearly from 0.1 t o 1.0 M) f r o m one column t o the next
within six days. Each trinucleotide fraction contained 100 t o
300 mg. They were diluted tenfold with H z O a n d purified
a n d desalted in fou r stages b y sorption on DEAE-cellulose
filters of decreasing diameter (each 4 cm in height) an d desorption with about 1 M am m o n iu m hydrogen carbonate
( p H 7.8). T he solutions of oligonucleotides were concentrated, acidified with cellulose phosphate (P-cellulose, p. a.,
from Serva. Heidelberg), filtered, centrifuged, a n d lyophilized.
T h e sequence of each trinucleotide was determined by
high-voltage paper electrophoresis of th e products obtained
590
f r o m i t by hydrolysis either at 37OC with 0.5 N K O H or
with TI-RNase.
75 mg of A p G p C p (or 60 mg of A p G p U p j were dissolved by
ultrasonic agitation in 1.1 (0.9) g of ethyl tetrametaphosphate
[4a] plus 0.125 (0.10) ml of anhydrous trisdimethylamino
phosphate an d polymerized by rotating at 60°C for 48 h in
a sealed tube [4bl. Dialysis of the products overnight gave
a loss of 44 % (45 %) of the ultraviolet-absorbing material.
Th e solution retained by the dialysis membrane was concentrated a n d separated o n a column (3 c m in diameter,
150 cm long) of Sephadex (3-75 into fractions of various
chain-lengths.
Th e coding properties of fractions with a chain length of
ab o u t 18 nucleotides was investigated using o u r previously
published method [ 5 ] ; however, for t h e purpose of peptide
synthesis, a complete protein-synthetizing system [6], including t h e supernatant from I00000 g centrifugation of
a n almost ribonuclease-free extract of Eschericlzirr coii, was
used. T h e mixtures were incubated for 3 min at 0 “ C a n d then
For 7 mi n at 36 “C. Most of the experiments with poly-ApGpUp
a n d poly-ApGpCp gave a specific increase i n the incorporation of asparagine. I n some experiments with poly-ApGpUp
also methionine was incorporated, but this was presumably
d u e t o cleavage of the terminal nucleotide. These findings
agree well with the observation that the trinucleotides A p G p C
a n d A p G p U , which have been freed from the 3’-terminal
phosphate, effect specific attachment of asparaginyl-RNA
o n t o ribosomes following t h e method described i n t he
previous communication [5].
T h e polynucleotide-dependent incorporation was ab o u t 20
times as high in the complete protein synthetizing system as
in the simplified system for amino acid adaptation [2,5]. The
peptides presumably formed will be investigated.
Received, August 4th, 1964
[Z 793/602 [El
German version: Angew. Chem. 76, 717 (1964)
__
~~
[*I We are indebted to Misszs F. Schurnbrand and
K . Eckert for
valuable assistance.
[ I ] J . H. Matthaei, Nova Acta Leopoldina N. F. 26, 45 (1963).
[2] J. H. Matfhaei, Lecture at the Bunsen-Tagung, Berlin, May
1964.
[3] M. W. Nivenherg, recently reported similar results at the VIth
International Congress of Biochemistry, New York, J~i l y1964.
[4a] W. Pollmann and G. Schramm, Biochim. biophysica Acta
80, I (1964).
[4b] G. Schramni, H. Grotsch, and W. Pollmann, Angew. Chem.
74, 53 (1962); Angew. Chem. internat. Edit. I , I (1962).
[5] F. Cramer, H . Kuntzel, and J. 17.
Mafthaei, Angew. Chem. 76,
716 (1964); Angew. Chem. internat. Edit. 3, 589 (1964).
[6] J . H . Matthaei and M. W. Nirenberg, Proc. nal. Acad. Sci.
USA 47, 1580 (1961).
A New Type of Phosphorus-Nitrogen Compound
with Pentavalent Phosphorus
By Prof. Dr. M. Becke-Goehring a n d L. Leichner
Anorganisch-chemisches Institut der Universitiit Heidelberg
(Germany)
Following t h e interaction of PC l j with amines, products of
type ( I ) or (2) have previously been obtained [I]. C o mp o u n d
(2) is formed in t h e reaction of [(CH3)NHj]CI with PC15. If
methylammonium chloride containing a few percent of water
(2)
R = CH,
-
R- N-P-N-
I
H
ci ‘c1
i3j
Angew. Chem. internat. Edit.
1 Vol. 3 (1964)
No. 8
Документ
Категория
Без категории
Просмотров
1
Размер файла
263 Кб
Теги
sequence, determination, triple, code
1/--страниц
Пожаловаться на содержимое документа