Vol. 123, No. 2 Printed in U.S.A.

JOURNAL OF BACTERIOLOGY, Aug. 1975, p. 765-767 Copyright 0 1975 American Society for Microbiology

Characterization of Polyadenylate from the Fungus Trichoderma viride DALIA ROSEN,* MARVIN EDELMAN, AND ESRA GALUN Department of Plant Genetics, Weizmann Institute of Science, Rehovot, Israel Received for publication 5 May 1975

Polyribonucleotide segments, about 60 nucleotides long and consisting of about 95% adenylic acid residues, were isolated from whole cell ribonucleic acid of the deuteromyceteous fungus Trichoderma viride. Similar findings in two other groups of the true fungi raise the possibility that short polyadenylate sequences may be a feature of these relatively simple organisms.

Polyadenylate [poly(A) ]-containing messenger (ribonucleic acid) RNA species have been found in eukaryotes ranging from mammals (3, 13), vascular plants and algae (19) to slime molds (4) and true fungi (12). Poly(A) sequences have also been demonstrated in viruses (16) and in mitochondria of mammals and insects (8). The length of poly(A) segments in messenger RNA molecules from various organisms ranges from 35 to 250 nucleotides. The biological significance of this size variation is not yet clear. Although short segments have been found attached to a few specific messenger RNAs in certain higher organisms (7, 9), generally speaking, the average nucleotide length of the poly(A) population within an organism seems to increase with increasing genome complexity (5, 20). The fungi have classically been considered to be among the simplest eukaryotes. Their haploid genome is quite small, being only about 2 to 10 times more complex than the average bacterial genome (21). Likewise, large heterogenous nuclear RNA could not be detected in these organisms (6). As part of an investigation of differentiation during conidial germination in Trichoderma viride (18), we have isolated and characterized poly(A) sequences from whole cell RNA extracted from conidia labeled with 32P1 or [14C Jadenosine. We wish to report here that the average poly(A) sequence of this hyphal fungus is about 60 nucleotides long. T. viride conidia were germinated as previously described (18). Nineteen hours after inoculation, the germinated conidia were labeled for 15 to 20 min with 0.1 mCi "2P1 or 0.2 ACi of [14C ]adenosine per ml of medium. RNA extraction and poly(A) isolation were carried out by two different methods: (i) RNA of cells labeled 15 min with radioactive precursor was extracted with tri-isopropyl-naphthalene-sulfonate-

phenol as described by Loening (10). Deoxyribonuclease treatment, pancreatic, and T1 ribonucleases digestion of the whole cell RNA, and poly(A) isolation by binding to and eluting from cellulose nitrate membrane filters (Millipore Corp.) (repeated twice), were as described by Sagher et al. (19). (ii) RNA of cells labeled 20 min with radioactive precursor was extracted with sodium dodecyl sulfate-hot phenolchloroform as described by McLaughlin et al. (cf. 12 and references therein). Deoxyribonuclease and ribonucleases treatments were performed as in (i) followed by isolation of poly(A) by oligodeoxythymidylate (oligo-dT) cellulose chromatography according to Aviv et al. (1). The final products obtained by the two methods were then characterized as to their nucleotide composition, electrophoretic mobility on polyacrylamide gels, and sedimentation through sucrose gradients as previously described (19). Figure 1 shows the 32P1-nucleotide composition of the ribonuclease-resistant and Millipore membrane bound RNA fraction [method (i)]. As can be seen, more than 95% of the radioactivity associated with the ribonucleotides co-electrophoresed with adenylic acid. Similar results were obtained by method (ii) (C = 0.1%, A = 94.6%, G = 4.6%, U = 0.7%). This fraction represents about 0.3 to 0.5% of the total radioactivity in the newly synthesized RNA. To determine the average size of the radioactive adenylate segments, the ribonucleaseresistant fraction was analyzed by both polyacrylamide gel electrophoresis and sedimentation velocity through sucrose with various marker RNAs. We observed that about 80% of the total poly(A) sequences of Trichoderma RNA obtained by oligo-dT or membrane filtration migrated on a gel to a position between 4S and 5S Escherichia coli RNA (Fig. 2A); these same sequences sedimented considerably slower 765

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marker RNAs are not unexpected (cf. 19) and have been reported by other investigators (5, 17, 19). Results similar to those shown in Fig. 2A were also obtained using germinated conidia labeled with [4C ]adenosine. Based upon the two methods applied, as well as the positions of other poly(A) samples relative to the same or similar RNA markers (15, 19), we estimate the poly(A) of Trichoderma to be about 60 nucleotides long. It should be noted that since poly(A) isolation was from rapidly labeled, whole cell RNA and not from polysomes, it may well be that our poly(A) preparaDistance migrated (cm) tion is a mixture of nuclear and cytoplasmic FIG. 1. 32Pi-nucleotide composition of the ribonu- RNA. While poly(A) segments are usually reclease-resistant and millipore membrane bound RNA ferred to as being attached to the 3' end of the fraction. The sample was hydrolyzed in alkali (0.3 M messenger RNA molecule, some internally tranKOH at 37 C for 18 h) followed by high-voltage paper scribed sequences (14) may also be present in electrophoresis (2). The four 3'-ribonucleotides were used as markers and their positions were deter- such preparations. Our data for the deuteromyceteous fungus mined by ultraviolet absorbance (dotted circles). The electropherogram was cut into 1-cm strips Trichoderma complement those obtained for which were counted in a toluene-based scintillation two other true fungi of different systematic groups: Saccharomyces (12, 17) and Achlya liquid. (20). In all three cases poly(A) segments 40 to 60 nucleotides long were found which accounted for a large percentage of the poly(A) in these cells. It is possible that short poly(A) segments will be found to be a general feature of fungal a~~~~~~~~~~~~~~~~~.C _ a 4 messenger RNAs, conceivably reflecting the genome (21) of these organisms. simple ~~~~~~~~0 x

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LITERATURE CITED

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5

15

25

Froction number

FIG. 2. (A) Electrophoretic mobility of 32Pi-labeled Trichoderma poly(A) obtained by membrane filtration [method (i)]. Electrophoresis was carried out in 9-cm long, 7.5% polyacrylamide gels for 75 min at 9 mA/gel in E Buffer containing 0.2%o sodium dodecyl sulfate (11). The distribution of 32P, radioactivity was determined in 2-mm gel slices by Cerenkov counting. The positions of 9S rabbit globin messenger RNA, 5S E. coli ribosomal RNA, and 4S E. coli transfer RNA were

determined by ultraviolet absorbance. 32Pi-

labeled poly(A) from the alga Euglena gracilis (obtained as described in 19) was run in a parallel gel under identical conditions. (B) Sucrose gradient distribution of 32Pi-labeled poly(A) isolated by oligo-dT cellulose chromatography [method (ii)]. The procedure of Sagher et al. (19) was followed. Marker RNAs were centrifuged in separate tubes. than E. coli 4S RNA on a sucrose gradient (Fig. 2B). In contrast, poly(A) from the alga Euglena

migrated on gels between 5S and 9S marker RNAs and sedimented in sucrose with 4S RNA. Differences between poly(A) sedimentation and electrophoretic patterns relative to specific

1. Aviv, H., and P. Leder. 1972. Purification of biologically active globin messenger RNA by chromatography on oligothymidylic acid-cellulose. Proc. Natl. Acad. Sci.

U.S.A. 69:1408-1412. 2. Edelman, M., I. M. Verma, and U. Z. Littauer. 1970. Mitochondrial rRNA from Aspergillus nidulkns: characterization of a novel molecular species. J. Mol. Biol.

49:67-83. 3. Edmonds, M., M. H. Vaughan, and H. Nakazato. 1971. Polyadenylic acid sequences in the heterogeneous nuclear RNA and rapidly-labeled polyribosomal RNA of HeLa cells: possible evidence for a precursor relationship. Proc. Natl. Acad. Sci. U.S.A. 68:1336-1340. 4. Firtel, R. A., A. Jacobson, and J. F. Lodish. 1972. Isolation and hybridization kinetics of messenger RNA from Dictyostelium discoideum. Nature (London) New Biol. 239:225-228. 5. Foquet, H., R. Bohme, R. Wick, H. W. Sauer, and R. Braun. 1974. Isolation of adenylate-rich RNA from Physarum polycephalum. Biochim. Biophys. Acta

353:313-322. 6. Gamow, E., and D. M. Prescott. 1972. Characterization of the RNA synthesized by Phycomyces blakesleeanus. Biochim. Biophys. Acta 259:223-227. 7. Gorski, J., M. R. Morrison, C. G. Merkel, and J. B. Lingrel. 1974. Size heterogeneity of polyadenylate sequences in mouse globin messenger RNA. J. Mol. Biol. 86:363-371. 8. Hirsch, M., A. Spradling, and S. Penman. 1974. The messenger-like poly(A)-containing RNA species from the mitochondria of mammals and insects. Cell 1:31-35.

VOL. 123, 1975 9. Lavers, G. C., J. H. Chen. and A. Spector. 1974. The presence of polyriboadenylic acid sequences in calf lens messenger RNA. J. Mol. Biol. 82:15-25. 10. Loening, U. E. 1967. The fractionation of high molecular weight RNA by polyacrylamide gel electrophoresis. Biochem. J. 102:251-257. 11. Loening, U. E. 1969. The determination of molecular weight of RNA by polyacrylamide gel electrophoresis. The effects of changes in conformation. Biochem. J. 113:131-138. 12. McLaughlin, C. S., J. R. Warner, M. Edmonds, H. Nakazato, and M. H. Vaughan. 1973. Polyadenylic acid sequences in yeast messenger ribonucleic acid. J. Biol. Chem. 248:1466-1471. 13. Mendecki, J., S. Y. Lee, and G. Brawerman. 1972. Characteristics of the polyadenylic acid segment associated with messenger ribonucleic acid in mouse sarcoma 180 ascites cells. Biochemistry 11:792-798. 14. Nakazato, H., M. Edmonds, and D. W. Kopp. 1974. Differential metabolism of large and small poly(A) sequences in the heterogeneous nuclear RNA of HeLa cells. Proc. Natl. Acad. Sci. U.S.A. 71:200-204. 15. Pemberton, R. E., and C. Balgioni. 1972. Duck hemoglo-

NOTES

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bin messenger RNA contains a polynucleotide sequence rich in adenylic acid. J. Mol. Biol. 65:531-535. Philipson, L., R. Wall, G. Glickman, and J. E. Darnell. 1971. Addition of polyadenylate sequences to virusspecific RNA during adenovirus replication. Proc. Natl. Acad. Sci. U.S.A. 68:2806-2809. Reed, J., and E. Wintersberger. 1973. Adenylic acid-rich sequences in messenger RNA from yeast polysomes. FEBS Lett. 32:213-217. Rosen, D., M. Edelman, E. Galun, and D. Danon. 1974. Biogenesis of mitochondria in Trichoderma viride: structural changes in mitochondria and other cell constituents during conidial maturation and germination. J. Gen. Microbiol. 83:31-49. Sagher, D., M. Edelman, and K. M. Jakob. 1974. Poly(A)-associated RNA in plants. Biochim. Biophys. Acta 349:32-38. Silver, J. C., and P. A. Horgen. 1974. Hormonal regulation of presumptive mRNA in the fungus Achlya ambisexualis. Nature (London) 249:252-254. Stork, R. 1974. Molecular mycology, p. 423-477. In J. B. G. Kwapinski (ed)., Molecular microbiology. John Wiley and Sons, Inc., New York.

Characterization of polyadenylate from the fungus Trichoderma viride.

Polyribonucleotide segments, about 60 nucleotides long and consisting of about 95% adenylic acid residues, were isolated from whole cell ribonucleic a...
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