Nucleic acid of insects
by
JOHN LEWIS ROSEDALE
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Division of Entomology, Pretoria
It has already been shown (Rosedale, 1945) that during metabolisism, insect nucleic acid is broken down to its components.
The nucleotide guanylic acid, which is concerned with an aminoacid deaminase system, was found to be one of the breakdown
products (Rosedale, 1946). Opportunity has now been taken of
extending the work. The subjects concerned in the present report
are soldiers and workers of the termite Trinervitermes havilandi,
though in one experiment the larvae of the blow-fly Chrysomyia
chloropyga Wied. were used. The pyrimidine bases have also been
isolated from a small sample of the fruit beetle, Pachnoda sinuata F.
The method employed was mainly that described by Brown and
Johnson (1923). Guanylic acid was found, as stated in the previous
papers (Rosedale, 1945, 1946), in the filtrate from the first precipitation of the nucleic acid. Guanylic acid was removed from this
filtrate as a lead salt and purified as a barium salt. A significant
amount of nitrogen remained. Tests for purines were negative but
the colour tests of Baudisch and Johnson (1922, 1923) and of Johnson
and Harkins (1929) indicated the presence of thymine. The solution
was treated with phosphotungstic acid which precipitated the nitrogenous compound. After removal of the phosphotungstic acid and
excess barium the compound gave on analysis 10.99 per cent of
nitrogen and 8.09 per cent phosphorus. On hydrolysis with acid the
products were found to be a nitrogenous base containing 33.6 per
cent nitrogen, phosphoric acid, and a sugar. The base reacted with
nitrous acid to form thymine. The base thus appeared to be methylcytosine, and the nucleotide from which it was derived methylcytidylic acid. Another sample of the nucleotide was trE:ated with
nitrous acid prior to acid hydrolysis. Thereafter the usual colour
tests for thymine were carried out and thymine shown to be present.
Another sample of 5-methyl-cytidylic acid was hydrolysed for 10
hours with 25 per cent sulphuric add. After quantitative removal
of the sulphuric acid, the solution was concentrated to a small
volume and the base precipitated with mercuric chloride. The precipitate was freed from mercury and the resulting solution concentrated further. A crop of small crystals were obtained which melted
with effervesence at 270°C and contained 33.6 per cent total nitrogen
and 11.1 per cent of amino nitrogen. After treatment with nitrous
35
Rosedale: NILcleic acid of insects
acid some crystals of thymine could be obtained which gave the
usual colour tests. It is thus established that the base of this
nucleotide is 5-methyl-cytosine and that the nucleotide is 5-methylcytidylic acid.
As stated above, 5-methyl-cytidylic acid together with' guanylic
acid has been found in the filtrate from the first precipitation of nucleic acid by the method of Brown and Johnson (1923).
The small residue or precipitation from this filtration was dissolved
in 2 per cent ammonia and heated on a water bath under a reflux
for 5 hours. To the filtrate, an equal volume of 95 per cent alcohol
was added and the small amount of residue filtered off proved to
be mainly phosphates. Brucine salts of adenylic acid and of cyUdylic
acid were prepared from the filtrate in accordance with the method
of Levene (1918, 1919). From these brucine salts the pure nucleotides
were obtained.
From an original weight of 337 grams of dry defatted termites
the yield of the nucleotides were:
Reproduced by Sabinet Gateway under licence granted by the Publisher (dated 2009).
Guanylic acid .,. ... ... ... ........
Adenylic acid ... ... ... ... ... ... ...
Cytidylic acid ... ... ..... ... ... ...
5-Methyl-cytidylic acid ...............
1.0657
1.1243
0.9602
0.9966
grams
grams
grams
grams
No free uracil nor thymine nor their nucleotides could be
obtained.
The above work was repeated using 568 grams of dry defatted
larvae of the blow-fly C. chrysomyia. Yields of nucleotides obtained
were:
Guanylic acid ..................... '"
Adenylic acid ............... '" .........
Cytidylic acid ... ... ... ... .., ... ... ...
5-Methyl-cytidylic acid ...............
1.8002
1.8951
1.6183
1.6801
grams
grams
grams
grams
A quantity of 26.2 grams of the dry defatted fruit beetle became
available, but owing to the limited quantity it was impossible to
prepare nucleotides, and accordingly, the material was hydrolysed
direct with 4 per cent sulphuric acid for 2 hours at 125°C. After
cooling and dilution the solution was treated with silver salts and
the precipitate filtered off and discarded. The filtrate was neutralised
with baryta, and a precipitate of pyrimidine bases obtained. This
was freed from silver and the resulting solution precipitated
with phosphotungstic acid. After regeneration of the phosphotungstates with baryta, cytosine M.P. 321 "C and 5-methyl-cytosine M.P.
270°C crystallised out.
343 grams of dry defatted termites which had been killed with
dichloro-diphenol-trichloroethane (D.D.T.) were then studied and
nucleotides prepared. The yields of nucleotides were:
36
Journal Ent. Soc. S. Africa. Vol. XI. 30th September, 1948.
Guanylic acid ... ... ... ... ..... '" ... 1.0846 grams
Cytidylic acid ........................ 0.9773 grams
5-Methyl-cytidine-di-phosphoric-ester 1.2421 grams
No adenylic acid could be found, but a search for adenine yielded 0.3512 grams of the. free base.
On hydrolysis, the 5-methyl-cytidine compound gave 0.1801
grams phosphorus (14.5%), instead of 8.09 per cent. Thus the product is 5-methyl-cytidine-di-phosphoric-ester. The excess phosphorus
over that required by 5-methyl-cytidylic-(mono)-phosphoric acid is
0.07962 grams, which in terms of adenylic acid would correspond to
0.3465 grams of adenine. This is within 1.33 percent of the amount
of adenine (0.3512 grams) found.
Accordingly it appears that adenylic acid has been broken down
by the D.D.T. and that the phosphorus therefrom has been taken up
quantitatively by the 5-methyl-cytidylic acid to form 5-methylcytidine-di-phosphoric-ester. Thus 5-methyl-cytidylic acid appears
to act as phosphorus acceptor.
Reproduced by Sabinet Gateway under licence granted by the Publisher (dated 2009).
Discussion:
The nucleotide 5-methyl-cytidylic acid has been found in termites and blow-fly larvae. It has not previously been found in nature,'
althou~h the base 5-methyl-cytosine was found by Johnson and
Coghill (1925) in tuberculinic acid. 5-methyl-cytosine has been found
in Pachnoda sinuata F. Thymine has not been found in any of the
three insects studied, and it was also not discovered among the
products of locust nucleic acid (Rosedale, 1945). It seems clear therefore that thymine is a deaminated product of the parent substance
5-methyl-cytosine.
N-CNH.,
HN-CO
I I - by deaminisation I I
OC
CCH,
I II
HN-CH
OC
CCH"
I
II
HN-CH
Thymine.
5-methyl-cytosine
It has been shown that under the influence of D.D.T. adenylic
acid is broken down and that its phosphate moiety is quantitatively
"accepted" by 5-methyl-cytidylic acid forming 5-methyl-cytidinedi-phosphoric-ester.
The physiological significance appears to be that D.D.T. interferes with the metabolism of carbohydrate. The first step in the
utilisation of carbohydrate by muscular tissue is the formation from
,lZlycogen 0f hexose-phosphate. Adenylpyrophosphate, under the influence of a hexosekinase is responsible for the formation of hexose
phosphate. Since adenylic acid loses its phosphate under the
influence of D.D.T. it seems clear that the ordinary system of
carbohydrate metabolism is upset.
,
1
1
j
~
I
Rosedale: Nucleic acid oj insects
37
Grateful acknowledgment is made to Mr. B. K. Petty for procuring the termites and spraying them with D.D.T. (3.5 per cent
para-para isomer) in kerosene.
Summary:
1. The nucleotides of T. havilandi, and the larvae of C. chloro-
pyga, have been isolated and are found to be guanylic acid, adenyiic
acid, cytidylic acid, and 5-methyl-cytidylic acid only.
2. When the termites are killed with D.D.T. guanylic acid and
cytidylic acid are still found to be present, but the 5-methyl-cytidylic
acid has been changed to 5-methyl-cytidine-di-phosphoric-ester, and
it was impossible to isolate any adenylic acid.
3. The free base adenine has been recovered to the amount
corresponding to that expected in termites which have not been
treated with D.D.T. and it appears that the phosphorus of the adenylic acid has been taken up quantitatively by the 5-methyl-cytidylic
acid to form 5-methyl-cytidine-di-phosphoric-ester.
4. Uracil and thymine could not be found in these insects and
it is considered that methyl-cytosine is the parent substance of
thymine.
Reproduced by Sabinet Gateway under licence granted by the Publisher (dated 2009).
References:
Baudisch and Johnson (1922): Ber. d. Deutsch. Chern. Ges. 55. 18.
Baudisch and Johnson (1923): J. Am. Chern. Soc. 43. 2670.
Brown and Johnson (1923): J. BioI. Chern. 57. 199.
Johnson and Coghill (1925): J. Am. Chern. Soc. 47. 2838.
Johnson and Harkins (1929): J. Am. Chern. Soc. 51. 1237.
Levene (1918): J. BioI. Chern. 33. 425.
Levene (1919): J. BioI. Chern. 40. 415.
Rosedale (945): J. S.A. Chern. Inst. 28. :t
Rosedale (1946): J. Ent. Soc. S.A. 9. 89.