Isolation and characterisation of nuv11, a mutation affecting meiotic and mitotic recombination in Aspergillus nidulans

A mutant of Aspergillus nidulans, designated nuv11, has been isolated as hypersensitive to the monofunctional alkylating agent MNNG and the quasi-UV-mimetic mutagen 4-NQO. The mutation was recessive, resulting from mutation of a single gene which mapped to chromosome IV, and was non-allelic to the previously characterised repair-deficient mutations uvsB and uvsH which are also located on this linkage group. The nuv11 mutation results in slow growth, deficient intragenic and intergenic meiotic recombination, increased spontaneous chromosome instability, and increased intragenic and intergenic mitotic recombination in homozygous diploids. By screening a wild-type gene bank of A nidulans, a clone (pNUV11A40) has been isolated which complements the nuv11 mutation, restoring wild-type responses to both MNNG and 4-NQO.     

Mutants of Aspergillus nidulans with increased resistance to the alkylating agent, N-methyl-N'-nitro-N-nitrosoguanidine

The isolation and characterisation of mutants of Aspergillus nidulans showing resistance to MNNG is described. Such isolates were stable through prolonged subculture in the absence of the selective agent, and resistance segregated as an allele of a single gene in meiotic and mitotic analysis. MNNG-resistant strains showed an increase in resistance to EMS and UV irradiation but no cross-resistance to MMS was detected. Possible mechanisms of resistance to alkylating agents are discussed.     

Cross-linking studies with the uvrA and uvrB proteins of E. coli

The interactions of the uvrA and uvrB proteins with DNA have been investigated using a DNA-protein cross-linking technique. It is demonstrated that hydrolysis of ATP by the uvrA protein facilitates cross-linking of this protein to single-stranded DNA, whether the DNA is UV irradiated or not. In contrast, cross-linking to unirradiated double-stranded DNA is not facilitated by ATP hydrolysis and is in fact increased by the substitution of the non-hydrolysable analogue aTP gamma S for ATP. In the presence of ATP, a dose-dependent increase is observed in the amount of uvrA protein which can be cross-linked to UV-irradiated double-stranded DNA. Binding of uvrB protein to puvrA-DNA complexes has a stabilising effect and increases the number of complexes which can be cross-linked whether the substrate is single- or double-stranded DNA. We can find no evidence that ATP hydrolysis by uvrA protein results in unwinding of UV-damaged DNA.     

Effectiveness of anticonvulsants in mice exposed to mixed gamma-neutron radiations

The effectiveness of four anticonvulsants was tested in male CF1 mice exposed to 500-, 1000-, and 10,000-rad doses of mixed gamma-neutron radiations. Prevention of the hind leg extensor component of a maximal convulsion induced by electroshock was selected as the end point for effective anticonvulsant activity of diphenylhydantoin, phenobarbital, and mephenytoin. Prevention of convulsions induced by pentylenetetrazol was the end point of effective anticonvulsant activity of trimethadione. The effectiveness of the drugs was evaluated by comparing the ED50's to the ED50 value for unirradiated controls. The anticonvulsants tested by electroshock showed a tendency toward increasing effectiveness after 500- and 1000-rad doses of radiation and a significantly increased effectiveness following 10,000 rads. Trimethadione effectiveness in irradiated mice was similar to that in unirradiated controls at all doses and times tested.     

The spectra of base substitutions induced by the impCAB,mucAB and umuDC error-prone DNA repair operons differ following exposure to methyl methanesulfonate

We have used the lacZ reversion assay to study the mutation spectra induced by the Escherichia coli chromosomal umuDC operon and of its two plasmid-borne analogues impCAB and mucAB following exposure of cells to UV light and methyl methane-sulfonate (MMS). We have shown that the impCAB, mucAB and umuDC operons all produce a similar response to UV light which results almost exclusively in AT → GC transitions. However, we found that the three operons produced different responses to alkylating agents. We found that with MMS the chromosomal umuDC operon produced almost exclusively AT → GC transitions, whilst both mucAB and impCAB produced predominantly transversions. In the case of the impCAB operon the mutation spectrum contained more AT → TA than GC → TA transversions; this balance was reversed with mucAB. The effect of the copy number of the error-prone DNA repair operons upon the mutagenic spectra was also studied. The results obtained suggest that the copy number of the imp operon does not greatly affect the specificity of base substitutions observed. However, an increase in the copy number of the umuDC operon greatly affected the specificity of base substitution, such that virtually no transitions were produced and the spectrum was dominated by GC/AT → TA transversions. It appears that the three error-prone DNA repair operons impCAB, mucAB and umuDC, despite showing strong structural and functional homologies, can display major differences in the spectrum of base changes induced during mutagenesis. We propose that the type of misincorporation/chain extension which DNA polymerase III is allowed to synthesize on a damaged DNA template is extremely sensitive to both the amount and type of error-prone repair proteins present. The modulation of these events by the different proteins can result in widely different mutagenic changes in the repaired DNA.     

Volume regulation by flounder red blood cells in anisotonic media

The nucleated high K, low Na red blood cells of the winter flounder demonstrated a volume regulatory response subsequent to osmotic swelling or shrinkage. During volume regulation the net water flow was secondary to net inorganic cation flux. Volume regulation the net water flow was secondary to net inorganic cation flux. Volume regulation after osmotic swelling is referred to as regulatory volume decrease (RVD) and was characterized by net K and water loss. Since the electrochemical gradient for K is directed out of the cell there is no need to invoke active processes to explain RVD. When osmotically shrunken, the flounder erythrocyte demonstrated a regulatory volume increase (RVI) back toward control cell volume. The water movements characteristic of RVI were a consequence of net cellular NaCl and KCl uptake with Na accounting for 75 percent of the increase in intracellular cation content. Since the Na electrochemical gradient is directed into the cell, net Na uptake was the result of Na flux via dissipative pathways. The addition of 10(-4)M ouabain to suspensions of flounder erythrocytes was without effect upon net water movements during volume regulation. The presence of ouabain did however lead to a decreased ration of intracellular K:Na. Analysis of net Na and K fluxes in the presence and absence of ouabain led to the conclusion that Na and K fluxes via both conservative and dissipative pathways are increased in response to osmotic swelling or shrinkage. In addition, the Na and K flux rate through both pump and leak pathways decreased in a parallel fashion as cell volume was regulated. Taken as a whole, the Na and K movements through the flounder erythrocyte membrane demonstrated a functional dependence during volume regulation.     

Comparison of the nucleotide sequence of cloned DNA coding for an apolipoprotein (apoVLDL-II) from avian blood and the amino acid sequence of an egg-yolk protein (apovitellenin I): equivalence of the two sequences

We have compared the amino acid sequences of two low-molecular-weight avian apoproteins: apoVLDL-II from very low-density lipoproteins of hen plasma and apovitellenin I from hen egg yolk. The sequence of White Leghorn apoVLDL-II was derived from the nucleotide sequence of cloned apoVLDL-II DNA (Chan et al., 1980). The sequenator was used to determine the amino acid sequence of apovitellinin I from two breeds of hen (White Leghorn and Australorp). The sequences from the two breeds were not only identical, but they also completely matched the predicted sequence derived from the apoVLDL-II DNA sequence. The identity reported here establishes that this protein is transported intact from the blood to the egg yolk.     

Primary structure of kunitz-type trypsin inhibitor-2a (pI 5.9) fromPsophocarpus tetragonolobus (L.) DC seed

The primary structure of acidic trypsin inhibitor-2a (WBTI-2a,pI 5.9) fromPsophocarpus tetragonolobus (L.) DC seed was determined. This inhibitor consists of a single polypeptide chain of 180 amino acids including four half-cystine residues and has an N-terminal residue of pyroglutamic acid. The sequence of WBTI-2a,pI 5.9, showed 84% identity to acidic trypsin inhibitor-2 (WBTI-2,pI 5.1) but only 57% identity to the basic trypsin inhibitor (WBTI-1,pI 8.9) and 50% identity to the chymotrypsin inhibitor of winged bean. The data indicate that winged bean seed contains a family of three Kunitz-type inhibitors which have about 50% identity.