The occurrence of dihydro ribothymidine in chromosomal RNA

VoL.33,

No. 5, 1968

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

THE OCCURRENCEOF DIHYDRO RIBOTHYMIDINE IN CRROMOSOMAX, WA1 Ralph A, Jacobson2 andJames Banner Division California

of Biology

Institute

of Technology

Pasadena, California ReceivedOctober

9, 1968

Dihydrouridine midine

(hang,

(Madison and Halley,

et al.,

1968).

cRNAs would contain

of d.iHU to &alanine,

this

1957; Ceriotti

presence of a different

cRNA yields

reaction

that

spontaneously

pyrinidine this

chemical

a positive groups,

conversion

proof of the pre-

test

nucleotide

(Cline

as does di.HU.

in ret aacites

Thus,the

tumor cFINA is

and Shugar, 1960). to rat ascites

from the auto-analyzer

the conversion

et al.,

could be diHC, since this

of Magrath and Shaw when applied

acid,

have been assumed

unequivocal

to diHU (Janion

an amino acid which elutes

of 6-amino-isobutyric

in cbromo-

tumor cRNA does not produce any B-alanine

1963) for ureido

saturated

It is unlikely deaminates

a specific

even though it yields

and Spandrio,

mounts

pyrimidine.

a method which provides

under these conditions,

conversion

~cct,m~ tmiver&.ly

It might reasonably

sence of diHU in RNA. Rat ascites

readily

It

reduced pyri-

1965) and of chicken embryos

Magrath and Shaw (1967) have described

indicated.

occurring

1965).

of peas (Huang and Banner,

1967; hang all

naturally

RNAs, It has also been found in substantial

SOB&IL RNA (cIIIYA)

that

(diHU) was the first

to be discovered

in transfer

91109

anslog

of

dihydfo

The tumor

in the region ribothymidine

(WIT). This paper describes

experiments

which prove the existence

of diH!L'

1‘This work was supported in part by U. S. Public Health Service grant ~~-13762 and by a U. S. Public Health Service Postdoctoral Fellowship 5-F-i?-OM 25,935. University, Nornan, Okla. 2p resent Address: Chemistry Department, Oklahoma 716

BIOCHEMICAL

Vol. 33, No. 5, 1968

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

as a component of the cRNAof rat ascites tumor. Methods Preparation

of authentic

dihydro ribothymidine:

to diHT using rhodium or alumina catalyst Isolation

Ribothymidine was reduced

(Cohn and Doherty, 1956).

of diHT from cRNA: Chromatin was prep-a

from ascites tumor cells

by the method of Dshmus(1968) and from calf thymus by the method of Husng et a~. (1964).

cRNAlinked to its binding protein

(&?A * BP, Banner and

Husng, 1966) was dissociated from the chromatin by salt or was isolated directly from the nuclear material which is separated from chromatin during sucro8e gradient purification detail

of the latter.

elsewhere (Jacobson, 1968).

DEAE-cellulose,

are described in

The &MA ?r BP was chromatographed on

recovered snd digested with a mixture of Tl and pancreatic

FU?aseor hydrolyzed binding protein

These preparations

with NaOH. Chromatography on Biogel P-30 yielded the

attached to a

bond between nucleotiae

saturated nucleotide.

reB%dw

and protein was hydrolyzed

phate removed with alkaline

The covalent

by 0.1 N XC1 snd the phos-

phosphatase.

Chrapstography of diHT snd &iHU: Open and closed ring forms of both nucleosides were chromatographed on thin lwer nated-cellulose

using O.lN

plates of polyethanolinine-impreg-

formic acid as eluent (Husng et al.,

were also separated on S&Sblackribbon graphy using the 3 different

1968).

They

paper (#589) by ascending chromato-

solvent systems described by Cline et &.

(1959)

as well as 0.1 N formic acid. The standards were identified P-dimethyl

amino bencaldehyde solqtion

(Cline et &I.., 1959).

For maximum

nucleosides from cRNAwere eluted by 0.1 N HCl and their

SenSitiYity,

determined using the more sensitive (x.963)

by spraying with 0.5 N NaOHfollowed by

color reaction

of Ceriotti

presence

and SpaMrio

l

Conversion to S amino acids: Nucleosides were converted to their

respective

B amino acids by the method of Magrath ana Shm (1967) aa the products separated on an amino

ida analyzer.

For mBxim\lmseparation,

717

elution

with

Vol. 33, No. 5, 1968

BIOCHEMICAL

pH 3.25 citrate isobutyric present

buffer

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

was coutiuued

acid was then eluted as abyproduct

ofthe

until

B alanine

by pH 4.2 citrate

was eluted.

buffer.

0 Amino

Anmouia is also

cozmrsiou. Results

Table 1 shows the Rf values for the nucleosides systems.

Since the differences

nucleosides

solvent

for the open ring ribosides,

of the cRRAs were snalysed only in the closed ring form.

state

native

were minimsl

in the varioti

of cRIVAthey exist

the acid incubatiau, pyrimidine

essential

to the closed ring

in the open ring,

ureido positive

to cleave the RNA-protein (Jauion

the

In the

form, but

bond, reaunesls

the

and Shugar, 1960).

Table 1 Rivedues for diHUsnddiHT in both the closed and open ring form diHU (authentic and calf thymus) Ringstructure Solvent

diHT (authentic and rat smites tumor) closed apen

closed

system

tert-B&H,

MM, NR40H

.78

019

.62

925

tert-BuOH,

MEK, HCl

l 73

.88

.78

.86

set-BuOH, H29

.24

RCOORanpaper

09-l

.81

A9

.92

HCOOHonTLC

.80

.90

071

.92

Figure 1 shows the elution acid flwn the auto aualyser. extended uutil buffer

6 alanine

indicate

&?A is diHT while that

.l25

of B alenine

If the elutiou

is eluted,

making the difference The results

pattern

0

0

and 8 smiuo isobutyric

with the first

buffer

is not

both amino acids elute with the secoud

obscure. that the saturated

from calf t-us

nucleoside

frm

sscites

is diHU, as shcnm by the Rf

718

tumor value8

BIOCHEMICAL

Vol. 33, No. 5, 1968

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

. 3-

r---NH3 2-

Relative h Moles NH3

/B-NH,-

&olanine-

Isobutyric

Acid

I -

JL?/\ ptl

3.25

Elurnt

e

pH 4.4 Eluont

Time

pH 12 Elurnt

of Elution

Figure 1. A portion of au amino acid elution profile showing relative positions of &alanine, @iii iaobutyric ecid ana srmnonia(a byproduct of the cleavage of dihydro p&midines by acid). Table 2 Nature aa proportion of aihyaro pyrimiaines contained in chromosomalRNAs of varied organisms

cRNA

Pyrimiaine

%

pea bud

aim3

8.5l

f3 alanine

chicken embryo2

dilfu

9.6

B ahnine

calfthymus3

am

8.5

B slanine

ascites tumor

diHT

8.1

fl amino isobutyric acid

3% uang and Bonner

Conversion Product

(1965) report a higher figure. The present value is based on the mre accurate analytical methods now available.

*H-g

(196-i') .

3Shih (1967).

719

Vol. 33, No. 5, 1968

listed

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

in Table 1 and the smino acid analysis.

and amount of saturated pyrimidine

Table 2 summarizes the nature

ribosides contained in all

studied cRNAs.

Acknowledgements The authors wish to extend their Marjorie

Thomasfor their

skillful

sincere thanks to Ludia Brown snd

preparation

of chromatin, and to John

Rats for the amino acid analyses.

References Bonner, J. and Husng, R. C. C. SHistones (A.V.S. de Reuck and J. Knight, Eds.) Ciba Study Group 24, J. and A. Churchill, London, 1966, p. 18. Ceriotti, G. and Spandrio, L., Clin. Chim. Acta 8, 295 (1963). Cline, R. E., Fink, R. M., and Fink, K., J. Am. Cbem. Sot. 8l, 2521 (1959). Cohn, W. E., and Doherty, D., J. Am. Chem. Sot. 2, 2863 (1956). Dshmus,M. E., Ph. D. Thesis, California Institute of Technology, 1968. HUEUI~, R. c. c., Fed. Proc. iXi, 603 (196-f). Huaug, R. C. C., Banner, J., and Murray, K. J. Mol. Biol. 5, 54 (1964). Husng, R. C. C., and Bonner J., Proc. Natl. Acaa. Sci. U.S. &, 960 (1965). Huang, R. C, C., Alexander, E., and Huang, P. C., J. Mol. Biol., in press. Jacobson, R. A., in preparation. Janion, C., and Shugar, D., Acta Biocim. Polon. 1, 309 (1960). Madison, J. T., and Halley, R. W., Biochem. Biophys. Res. Corn. Is, 153 (1965). Magrath, D. I., end Shaw, D. C., Biochem. Biophys. Res. Comm.&, 32 (1967). Shih, T. Y., unpublished observation (1967).

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