Purification of Escherichia coli 30S ribosomal proteins by high performance liquid chromatography

High performance liquid chromatography was applied to the separation of proteins derived from the Escherichia coli 30S ribosomal subunit. Several methods of separating this protein mixture has been tested: size-exclusion chromatography on hydrophilic phases; ion exchange and reversed phase chromatography (on C2 to C18 hydrocarbon-bonded supports). Various elution systems were examined in order to obtain pure proteins suitable for micro-sequence analysis. The resolution and yields of the proteins varied considerably, depending on the type of support and gradient system used. The best results were achieved with uniformly globular-shaped supports of large pore size, and by combining high performance size exclusion with rechromatography on reversed phase columns. Purification conditions for the individual proteins are listed. The methods employed avoid any precipitation step and allow easy identification of the proteins by one or two-dimensional gel electrophoresis, amino-acid analysis or direct manual or automatic micro-sequencing. Since the isolation time is much reduced compared with conventional purification procedures, the proteins obtained by the techniques described here are well suited for topographical and immunological studies or reconstitution assays. Ribosomal proteins of other organisms can be separated under similar conditions.     

Temperature control of initiation of protein synthesis in Escherichia coli

When an exponentially growing culture of Escherichia coli is cooled to below 8 °C, initiation of protein synthesis appears to be blocked, while the elongation of initiated proteins continues until they are completed. This is demonstrated here by showing that nascent polypeptide chains increase in size during a 5 °C incubation and that f2 viral coat protein is completed, but not initiated. Upon rewarming, the cells initiate protein synthesis synchronously. This is demonstrated by a transient rise in the incorporation of methionine which is used to initiate protein synthesis.     

Genetic Recombination in Coprinus. IV. a Kinetic Study of the Temperature Effect on Recombination Frequency

At the restrictive conditions (35 degrees under continuous light) Coprinus lagopus is unable to initiate premeiotic S phase which takes place normally within 8-10 h of karyogamy. A shift-up to the restrictive conditions causes an arrest of the basidiocarps at this critical stage. A prolonged arrest causes a reversal to mitosis (Lu 1974b). Incubation of basidiocarps at the restrictive conditions before this critical stage causes no increase in recombination frequency (R.F.) in the loci studied. An arrest of 4 h at the critical stage still causes no R.F. increase, but 12-13 h and 18-19 h arrests cause increases of 50% and 90% over the controls, respectively. Thus R.F. can be increased even before the cells are fully committed to meiosis.-A 3-h heat treatment at the beginning of S phase (or 8 h before karyogamy) also causes some (30%) increase in R.F. while the same treatment at late S phase (or 3 h before karyogamy) causes a substantial (164%) increase in R.F. over the controls. A 3-h heat treatment before S phase causes no increase in R.F.-Pachytene is also responsive to temperature treatments (Lu 1969). The maximum R.f. increase is 100% by heat and 220% by cold treatment. The shortest time that can cause the maximum increase in recombination by high temperature is 3 h and that by cold treatment is 7 h. These durations are correlated with the length of the pachytene stage under the treatment conditions. The kinetic data show that the increase in R.F. caused by high and low temperatures follows two-hit kinetics and their rate of increase is almost identical. The higher increase in R.F. by low temperature can be attributed to the increased duration of pachytene and therefore R.F. is a function of time. The longer the homologous chromosomes are held together, the higher the recombination frequency.     

Conversion of sterigmatocystin to aflatoxin B 1 by Aspergillus parasiticus

¹⁴C-Sterigmatocystin isolated from cultures of $\text{Aspergillus}$$\text{versicolor}$ supplemented with (1-¹⁴C)acetate was shown to be efficiently converted to aflatoxin B₁ by the resting mycelium of $\text{A. parasiticus}$. The experimental results may indicate a biosynthetic pathway leading from 5-hydroxysterigmatocystin to sterigmatocystin and then to aflatoxin B₁.