Characterization of a temperature-sensitive mutant of Saccharomyces cerevisiae that undergoes uncontrolled protein synthesis.

A mutant of Saccharomyces cerevisiae, DW137, isolated after treatment of a wild-type strain with ICR-170. The mutant was respiration-deficient and showed abnormal cell division when grown at 30 degrees C. In addition, the mutant was temperature-sensitive and underwent lysis when grown at 37 degrees C. Random spore analysis, induced reversion profiles, and complementation analysis indicated that the abnormal phenotypes were under the control of a single recessive mutation caused by a base-pair substitution in a nuclear gene. Macromolecular analysis of the mutant at permissive and restrictive temperatures showed that at restrictive temperatures the mutant cannot synthesize DNA. Surprisingly, at restrictive temperatures, protein synthesis in the mutant continued at a rate greater than that observed at permissive temperatures. Cell death and lysis of the mutant could be prevented by treatment of cultures with cycloheximide, an inhibitor of protein synthesis. The data suggest that the abnormally high rate of protein synthesis and the inability to synthesize DNA are jointly responsible for death of the cells, and most probably play and integrating role in the incipient cell lysis.     

The attachment to, and invasion of HeLa cells by Salmonella typhimurium: the contribution of mannose-sensitive and mannose-resistant haemagglutinating activities

The association of the haemagglutinating activities of Salmonella typhimurium cultures with bacterial adhesion to HeLa cells, and the internalization of the bacteria by HeLa cells, was studied. Adhesion was not inhibited by alpha-methyl-D-mannoside (i.e. adhesion was mannose-resistant), and only four of the six strains tested produced type 1 fimbriae and the associated mannose-sensitive haemagglutinin (MSHA). The other two strains belonged to the non-fimbriate FIRN biogroup. Cultures of all six strains contained a mannose-resistant haemagglutinating (MRHA) activity when grown at 37 degrees C, but cultures of only one fimbriate and one non-fimbriate strain did so when grown at 18 degrees C. From the comparison of cultures grown at 18 degrees C and 37 degrees C, and of mutant strains with the phenotypes MRHA-negative/MSHA-positive, or MRHA-positive/MSHA-negative, it was concluded that the MRHA activity was responsible for the attachment of salmonellae to HeLa cells. Only bacterial adhesion that was resistant to mannose resulted in the internalization of the bacteria by the HeLa cells.     

Cell culture factors influencing in vitro expression of mouse mammary tumor virus.

Several cell culture factors were found to influence in vitro expression of mouse mammary tumor virus (MMTV) in the mouse adenocarcinoma cell line Mm5mt/c1. Cells were propagated in a variety of commercially available cell culture media to which dexamethasone (DXM) was added as a stimulator of MMTV production. Culture seeding density, culture medium type, and glucose concentration each influenced MMTV production when expressed on a per cell basis. Maximum cell growth occurred in cultures grown in RPMI-1640 medium containing insulin. Those media which provided good cell growth were not necessarily optimal for virus expression. Addition of insulin did not stimulate MMTV synthesis although dexamethasone alone was stimulatory in all media used; however, maximum MMTV expression was obtained when dexamethasone and insulin were used in concert. Equivalent levels of MMTV-specific cell membrane antigen, MMTV-specific protein, and virus particles were produced at incubation temperatures of 32 degrees, 34 degrees or 37 degrees C; however, higher levels of virus-related RNA-dependent DNA polymerase (RDDP) activity were recovered from cultures incubated at 32 degrees and 34 degrees C than at 37 degrees C. Decreased levels of RDDP were attributed to enzyme thermolability at 37 degrees C incubation.     

Escherichia coli Mutants Tolerant to Beta-Lactam Antibiotics

Two types of Escherichia coli mutants tolerant to beta-lactam antibiotics were isolated. One is E. coli chi2452, which showed a tolerant response against beta-lactam antibiotics when grown at 42 degrees C, and the others are the mutants C-80 and C-254, selected from mutagenized E. coli chi1776 by cycles of exposure to ampicillin, cephaloridine, and starvation of the nutritionally required diaminopimelic acid. Beta-lactam antibiotics caused rapid loss of viability and lysis in cultures of chi1776 or in chi2452 grown at 32 degrees C. In contrast, the same antibiotics caused only a reversible inhibition of growth in mutants C-80 and C-254 or in cultures of chi2452 grown at 42 degrees C. Beta-lactam antibiotics that show high affinity for penicillin-binding proteins 2 or 3 (mecillinam and cephalexin, respectively) induced similar morphological effects (ovoid cell formation and filament formation) in both parent and mutant strains. In contrast, beta-lactam antibiotics which have a high affinity for penicillin-binding protein 1 (e.g., cephaloridine or cefoxitin), which cause rapid lysis in the parental strains, caused cell elongation in the tolerant bacteria. In contrast to the parental cells, autolytic cell wall degradation was not triggered by beta-lactam treatment of chi2452 cells grown at 42 degrees C or in mutants C-80 and C-254. The total autolytic activity of mutants C-80 and C-254 was less than 30% that of the parent strain. However, virtually identical autolytic activities were found in cells of chi2452 grown either at 42 or 32 degrees C. Possible mechanisms for the penicillin tolerance of E. coli are considered on the basis of these findings.     

Enhanced secretion of cell wall bound enolase into culture medium by the soo1-1 mutation of Saccharomyces cerevisiae

In order to identify the protein(s) secreted into culture medium by the soo1-1/ret1-1 mutation of Saccharomyces cerevisiae, proteins from the culture medium of cells grown at permissive (28 degrees C) and non-permissive temperatures (37 degrees C), were analyzed. Comparison of protein bands separated by SDS-PAGE identified a prominent band of 47-kDa band from a mutant grown at 37 degrees C. N-terminal amino acid sequencing of this 47-kDa protein showed high identity with enolases 1 and 2. Western blot analysis revealed that most of the cell wall-bound enolase was released into the culture medium of the mutant grown at 37 degrees C, some of which were separated as those with lower molecular weights. Our results, presented here, indicate the impairment of cell wall enolase biogenesis and assembly by the soo1-1/ret1-1 mutation of S. cerevisiae.     

Cell wall proteins of Candida albicans

Proteins were solubilized from cell wall fractions of Candida albicans and separated by polyacrylamide gel electrophoresis. Cell walls were isolated from 25 and 37 degrees C growing and stationary phase yeast cultures and from germ tubes. The 42 protein bands detected by dye binding were observed in all wall extracts, regardless of the temperature, growth state, or morphology of the culture. The carbohydrate content of most bands was below the detectable limit of the periodic acid Schiff reagent. The protein complement revealed by autoradiography of radiolabeled proteins was half that detected by staining. Two bands showed greater intensity from cultures grown at 37 degrees C. The radio-labeled pattern was similar with both [35S]methionine-and [14C]leucine-labeled proteins and either pulse- or continuous-labeled proteins.     

Temperature-sensitive Escherichia coli mutant with an altered initiation codon of the uncG gene for the H+-ATPase gamma subunit.

A mutant of Escherichia coli showing temperature-sensitive growth on succinate was isolated, and its mutation in the initiation codon (ATG to ATA) of the uncG gene (coding for the gamma subunit of H+-ATPase F0F1) was identified. This strain could grow on succinate as the sole carbon source at 25 and 30 degrees C, but not at 37 or 42 degrees C. When this strain was grown at 25 degrees C on succinate or glycerol, its membranes had about 15% of the ATPase activity of wild-type membranes, whereas when it was grown at 42 degrees C, its membranes had about 2% of the wild-type ATPase activity. Membranes of the mutant grown at 25 or 42 degrees C could bind F1 functionally, resulting in about 40% of the specific activity of wild-type membranes. The gamma subunit was identified in an EDTA extract of membranes of the mutant grown at 25 degrees C, but was barely detectable in the same amount of extract from the mutant grown at 42 degrees C. These results indicate that initiation of protein synthesis from the AUA codon is temperature sensitive and that the gamma subunit is essential for assembly of F1 in vivo as shown by in vitro reconstitution experiments (S. D. Dunn and M. Futai, J. Biol. Chem. 255:113-118, 1980).     

Adenosine triphosphatase of Aspergillus nidulans: existence of isoenzymes of Ca2+-ATPase.

Ca2+-ATPase activity increased five- to six fold when the cells were subjected to growth at 37 degrees C in protein hydrolysate-supplemented media as compared to that of the cells grown in minimal media. One major isoenzyme and one minor isoenzyme were present in minimal-medium-grown cells while two major isoenzymes were present in the cells grown in protein-supplemented media. When the cells were subjected to heat stress (43 degrees C), they exhibited significantly decreased activity as compared to 37 degrees C grown cells. However, all the cultures subjected to growth at 43 degrees C showed two isoenzymes independent of growth medium.     

High temperature induced antibiotic sensitivity changes in Pseudomonas aeruginosa.

Pseudomonas aeruginosa, which was resistant to a wide variety of antibiotics, became sensitive to several of these antibiotics when grown and tested at 46 degrees C. Cell wall antibiotics such as penicillin G and ampicillin were only effective when added to cells growing at 46 degrees C prior to a temperature shift to 37 degrees C. Antibiotics which penetrate the cytoplasmic membrane to express their inhibiting action present a pattern different from those which are active against the outer cell wall. In order that these compounds be effective, the permeability of the cytoplasmic membrane must be further altered with agents such as EDTA which allow the penetration of actinomycin D. Inhibitors of protein synthesis, such as streptomycin and chloramphenicol, have increased access to their sites of action in cells grown at 46 degrees C. Cells grown at 46 degrees C have 40% less lipopolysaccharide (LPS) than cells grown at 37 degrees C and the LPS aggregates were of large molecular size in cells grown at 46 degrees C. Growth at 46 degrees C affects the permeability properties of the outer cell wall more than the permeability properties of the cytoplasmic membrane and this was due, in part, to the selective release of LPS of LPS-protein complexes at elevated growth temperatures.     

Isolation and characterization of Schizosaccharomyces pombe mutants defective in cell wall (1-3)beta-D-glucan.

Schizosaccharomyces pombe thermosensitive mutants requiring the presence of an osmotic stabilizer to survive and grow at a nonpermissive temperature were isolated. The mutants were genetically and biochemically characterized. In all of them, the phenotype segregated in Mendelian fashion as a single gene which coded for a recessive character. Fourteen loci were defined by complementation analysis. Studies of cell wall composition showed a reduction in the amount of cell wall beta-glucan in three strains (JCR1, JCR5, and JCR10) when growing at 37 degrees C. Galactomannan was diminished in two others. Strains JCR1 and JCR5, with mutant alleles cwg1-1 and cwg2-1, respectively, were further studied. The cwg1 locus was mapped on the right arm of chromosome III, 18.06 centimorgans (cM) to the left of the ade5 marker; cwg2 was located on the left arm of chromosome I, 34.6 cM away from the aro5 marker. (1-3)beta-D-Glucan synthase activities from cwg1-1 and cwg2-1 mutant strains grown at 37 degrees C were diminished, as measured in vitro, compared with the wild-type strain; however, Km values and activation by GTP were similar to the wild-type values. Mutant synthases behaved like the wild-type enzyme in terms of thermostability. Analyses of round shape, lytic behavior, and low (1-3)beta-D-glucan synthase activity in cultures derived from ascospores of the same tetrad showed cosegregation of all these characters. Detergent dissociation of (1-3)beta-D-glucan synthase into soluble and particulate fractions and subsequent reconstitution demonstrated that the cwg1-1 mutant was affected in the particulate fraction of the enzymatic activity while cwg2-1 was affected in the soluble component. The antifungal agents Papulacandin B and Aculeacin A had similar effects on the enzymatic activities of the wild type and the cwg2-1 mutant strain, whereas the cwg1-1 mutant, when growing at 37 degrees C, had a more inhibitor-resistant (1,3)beta-D-glucan synthase. It is concluded that the cwg1+ and cwg2+ genes are related to (1,3)beta-D-glucan biosynthesis.     

A Highly Thermosensitive and Permeable Mutant of the Marine Yeast Cryptococcus aureus G7a Potentially Useful for Single-Cell Protein Production and its Nutritive Components

The highly thermosensitive and permeable mutants are the mutants from which intracellular contents including proteins can be released when they are incubated both in the low osmolarity water and at the nonpermissive temperature (usually 37 degrees C). After mutagenesis by using nitrosoguanidine, a highly thermosensitive and permeable mutant named Z114 was obtained from the marine yeast Cryptococcus aureus G7a. Of the total protein, 65.3% was released from the mutant cells suspended in distilled water after they were treated at 37 degrees C overnight. However, only 12.3% of the total protein was released from the mutant cells suspended in 1.0 M sorbitol solution after they were treated at 37 degrees C overnight. We found that intracellular density of the mutant treated at 37 degrees C was greatly decreased, and cell volume of the mutant treated at 37 degrees C was increased due to the increased protein release. However, no significant changes in the intracellular density and cell volume of the mutant were observed when its cells suspended in 1.0 M sorbitol solution were treated at 37 degrees C. It was found that no big changes in cell growth, protein content, vitamin C content, nucleic acid content, fatty acids, and amino acid compositions of both the mutant and its wild type were detected. Therefore, the highly thermosensitive and permeable mutant still can be a good candidate as single-cell protein. This means that the method used in this study is a simple and efficient way to release protein from the highly thermosensitive and permeable yeast mutant cells with high protein content.     

Temperature-dependent switching between "wild-type" and "mutant" forms of p53-Val135

The p53 gene is a suppressor of abnormal cell growth but is also subject to oncogenic activation by mutation. The mutant allele p53-Val135, has recently been discovered to be temperature-sensitive and functions as an oncogene at 37 degrees C and as a tumor suppressor at 32.5 degrees C. In order to investigate the molecular mechanism underlying the temperature sensitivity of p53-Val135 rabbit reticulocyte lysate was used to translate the p53 mRNAs in vitro at 37 degrees C and at 30 degrees C. The immunoreactivity and T antigen binding of wild-type protein p53-Ala135 were unaffected by temperature and were similar to wild-type p53 expressed in vivo. In contrast, the mutant p53-Val135 protein was markedly affected by temperature. At 37 degrees C p53-Val135 showed reduced T antigen binding and did not react with monoclonal antibodies PAb246 and PAb1620. At 30 degrees C, p53-Val135 behaved as the wild-type p53. Temperature also exerted a post-translational effect on p53-Val135 with complete conversion from wild-type to mutant phenotype within two minutes of temperature shift from 30 degrees C to 37 degrees C. There was incomplete conversion from mutant to wild-type phenotype when the temperature was shifted down from 37 degrees C to 30 degrees C. We propose that the temperature dependent forms of p53-Val135 represent conformational variants of the p53 protein with opposing functions in cell growth control.     

Characterization of a thermosensitive sporulation mutant of Bacillus subtilis affected in the structural gene of an intracellular protease.

A thermosensitive sporulation mutant (ts-15) of Bacillus subtilis has been isolated. This mutant when grown at the restrictive temperature (42 degrees C) is unable to sporulate, shows no intracellular protease activity and no protein turnover. These three traits were recovered in two revertants (ts-15R1 and ts-15R2) and were also transmitted together by transformation into the wild type. Immunological studies have shown that when ts-15 is grown at 42 degrees C it synthesizes a 'cryptic' protein with apparently the same antigenic properties as the wild type or as ts-15 mutant grown at the permissive temperature (30 degrees C). The intracellular proteases from the wild type and from ts-15 grown at 30 degrees C and 42 degrees C were completely purified and their properties were studied with respect to their molecular weights, substrate specificity, inhibition pattern, heat inactivation and antigenicity. The molecular weight of the enzyme from the wild type or ts-15 grown at 30 degrees C was 64000--65000 in the absence of sodium dodecylsulfate and 31000--32000 in the presence of sodium dodecylsulfate. It was assumed therefore that the active enzyme is formed from two similar subunits. However, the intracellular protease from ts-15 grown at 42 degrees C showed the same molecular weight of 32000--34000 in the presence or in the absence of sodium dodecylsulfate. On the basis of this experiment and others described in the paper we concluded that the mutation in ts-15 is most likely a point mutation in a structural gene of an intracellular protease and results in an inability to assemble the two subunits into an active form.     

大肠杆菌发酵生产重组人源抗-HBs Fab

在KLF2000发酵罐中利用补料分批培养技术培养表达含重组质粒pBAD/HBs Fab的TOP10大肠杆菌,生产人源抗-HBs Fab,为批量生产作准备,在发酵过程中,控制溶氧30%以上,温度37℃,在基础培养基内生长4h后,补加以甘油为碳源的补料,继续生长到9h,加入阿拉伯糖,至终浓度为0.02%,30℃诱导表达5h,收集菌体,纯化制备目的蛋白。利用Western blot方法检测Fab抗原性,Dot blot方法检测生物学活性。14h发酵结束后,菌体密度最终达96g/L,纯化所得蛋白大约占菌体总蛋白的6%,含量为80mg/L,以重组质粒pBAD/HBs Fab,大肠杆菌TOP10表达表达比率与摇瓶相比没有降低,表达量达80mg/L左右,为大批量生产作了准备。     

Physiological studies of a temperature-sensitive sporulation mutant of Bacillus cereus T.

Growth of temperature-sensitive mutant Bacillus cereus T JS22-C occurred normally at the restrictive temperature (37 degrees C), but sporulation was blocked at stage 0. The production of extracellular and intracellular proteases and of alkaline phosphatase occurred at 37 degrees C, but the expression of a functional tricarboxylic acid cycle did not. At the permissive temperature (26 degrees C), the mutant sporulated at a slightly lower frequency (60%) and at a lower rate than the parent strain. The oxidation of organic acids, which accumulate in the growth medium began at T0 in cultures of the parent strain but was delayed until about T3 in cultures of the mutant. Later events in sporulation were also delayed in the mutant by about 3 h. Experiments in which the temperature of growth was shifted from 37 to 26 degrees C or from 26 to 37 degrees C at various times showed that the temperature-sensitive event began approximately 1 h after the end of exponential growth and ended when the cells reached the end of stage II (septum formation). The absence of a functional tricarboxylic acid cycle in cells of the mutant grown at 37 degrees C or shifted from 26 to 37 degrees C before T1 did not appear to be due to a lesion in one of the structural genes of the tricarboxylic acid cycle but was more likely due to the inability of the cells to derepress the synthesis of some of the enzymes of that cycle.     

Induction of Cr(VI) reduction activity in an Anoxybacillus strain under heat stress: a biochemical and proteomic study

A bacterial strain, designated as TSB-6, was isolated from the sediments of a Tantloi (India) hot spring at 65 °C. The strain showed 98% 16S rRNA gene sequence similarity with Anoxybacillus kualawohkensis strain KW12 and was found to grow optimally at 37 °C. However, growing cells, cell suspensions, and cell-free extracts from 65 °C cultures showed higher Cr(VI) reduction activities when assayed at either 37 or 65 °C than those obtained from 37 °C cultures. On fractionation of extracts from cells grown at 65 °C, the chromate reductase activity assayed at 65 °C was found mostly in the soluble fraction. When log-phase cells growing at 37 °C were shifted to 65 °C, the stressed cells produced larger quantities of reactive oxygen species. Consequently, growth of the cells was retarded, but specific Cr(VI) reduction activity increased. 2D gel electrophoresis followed by MALDI-TOF MS/MS identified the proteins whose expression level changed as a result of heat stress. The upregulated set included proteins involved in cellular metabolism of sugar, nucleotide, amino acids, lipids and vitamins, oxidoreductase activity, and protein folding. The downregulated proteins are also involved in cellular metabolism, DNA binding, and environmental signal processing.     

The BtpA protein stabilizes the reaction center proteins of photosystem I in the cyanobacterium Synechocystis sp. PCC 6803 at low temperature

Specific inhibition of photosystem I (PSI) was observed under low-temperature conditions in the cyanobacterium Synechocystis sp. strain PCC 6803. Growth at 20 degrees C caused inhibition of PSI activity and increased degradation of the PSI reaction center proteins PsaA and PsaB, while no significant changes were found in the level and activity of photosystem II (PSII). BtpA, a recently identified extrinsic thylakoid membrane protein, was found to be a necessary regulatory factor for stabilization of the PsaA and PsaB proteins under such low-temperature conditions. At normal growth temperature (30 degrees C), the BtpA protein was present in the cell, and its genetic deletion caused an increase in the degradation of the PSI reaction center proteins. However, growth of Synechocystis cells at 20 degrees C or shifting of cultures grown at 30 degrees C to 20 degrees C led to a rapid accumulation of the BtpA protein, presumably to stabilize the PSI complex, by lowering the rates of degradation of the PsaA and PsaB proteins. A btpA deletion mutant strain could not grow photoautotrophically at low temperature, and exhibited rapid degradation of the PSI complex after transfer of the cells from normal to low temperature.     

Organ culture of mammalian testis. III. Inhibin secretion.

Mouse testes were cultured for 19--20 days at either 31 or 37 degrees C with a change of medium every 4 days. After treatment with charcoal and dextran T, the recovered testis media were incubated with rat anterior pituitary cells, and secretions of follicle-stimulating hormone (FSH) and luteinizing hormone (LH) were estimated by radioimmunoassay 3 days later. FSH release was significantly lowered when pituitary cells were grown with media of testes cultured 31 degrees C compared to cultures grown with fresh medium or with media of testes cultured at 37 degrees C for more than 4 days. LH secretion was normal in one experiment and reduced in the other with the media of testes cultured at 31 degrees C. Treatment of testicular media by heat or trypsin reduced the inhibiting activity. After 8 days at 37 degrees C, both germinal and Sertoli cells were damaged in the testis cultures, while at 31 degrees germinal cells alone were destroyed, Sertoli cells remained normal. These studies suggest that (1) a substance which responds to the definition of inhibition (protein--preferentially acting on FSH) is secreted in the medium of testis culture; (2) inhibin is produced by Sertoli cells; (3) inhibin is secreted only if the temperature is inferior to 37 degrees C.     

Mutations in the tether region of the iron-sulfur protein affect the activity and assembly of the cytochrome bc(1) complex of yeast mitochondria

Resolution of the crystal structure of the mitochondrial cytochrome bc(1) complex has indicated that the extra-membranous extrinsic domain of the iron-sulfur protein containing the 2Fe2S cluster is connected by a tether to the transmembrane helix that anchors the iron-sulfur protein to the complex. To investigate the role of this tether in the cytochrome bc(1) complex, we have mutated the conserved amino acid residues Ala-86, Ala-90, Ala-92, Lys-93 and Glu-95 and constructed deletion mutants DeltaVLA(88-90) and DeltaAMA(90-92) and an insertion mutant I87AAA88 in the iron-sulfur protein of the yeast, Saccharomyces cerevisiae. In cells grown at 30 degrees C, enzymatic activities of the bc(1) complex were reduced 22-56% in mutants A86L, A90I, A92C, A92R and E95R, and the deletion mutants, DeltaVLA(88-90) and DeltaAMA(90-92), while activity of the insertion mutant was reduced 90%. No loss of cytochromes b or c-c(1), detected spectrally, or the iron-sulfur protein, determined by quantitative immunoblotting, was observed in these mutants with the exception of the mutants of Ala-92 in which the loss of activity paralleled a loss in the amount of the iron-sulfur protein. EPR spectroscopy revealed no changes in the iron-sulfur cluster of mutants A86L, A90I, A92R or the deletion mutant DeltaVLA(88-90). Greater losses of both protein and activity were observed in all of the mutants of Ala-92 as well as in A90F grown at 37 degrees C. suggesting that these conserved alanine residues may be involved in maintaining the stability of the iron-sulfur protein and its assembly into the bc(1) complex. By contrast, no significant loss of iron-sulfur protein was observed in the mutants of Ala-86 in cells grown at either 30 degrees C or 37 degrees C despite the 50-70% loss of enzymatic activity suggesting that Ala-86 may play a critical role in catalysis in the bc(1) complex.     

Characterization of RNA synthesis in an Escherichia coli mutant with a temperature-sensitive lesion in stable RNA synthesis

Previous experiments with Escherichia coli strain 2S142 have shown that the synthesis of stable RNA is preferentially blocked at the restrictive temperature. In this paper, we have examined the capacity of this mutant strain to synthesize RNA in vitro. Growth of the strain for as short a period as 10 min at 42 degrees C resulted in a 40 to 60% loss of RNA synthetic capacity and a fourfold decrease in percent rRNA synthesized in toluenized cell preparations. The time course for the loss and recovery of this RNA synthetic capacity correlated very well with the changes in RNA synthesis observed in vivo. We found no difference in temperature sensitivity of the purified RNA polymerase from the mutant and the parental strains. Moreover, there was no detectable alteration in the amount of enzyme, specific activity of the enzyme, or electrophoretic mobility of the subunits when the mutant strain was grown at 42 degrees C. The capacity for rRNA synthesis was also measured with the Zubay in vitro system (Reiness et al., Proc. Natl. Acad. Sci. 72:2881-2885, 1975). Supernatant fractions (S-30) prepared from cells grown at 30 degrees C were capable of up to 31.2% rRNA synthesis, using phi 80d3 DNA as template. S-30 fractions from cells grown at 42 degrees C synthesized 8.6% rRNA. The bottom one-third of the S-100 fraction and the ribosomal salt wash from 30 degrees C cells contained one or more factors which partially restored preferential rRNA synthesis in S-30 fractions from cells grown at 42 degrees C. Preliminary evidence suggests that the factor(s) is protein in nature.