Andrew A. Hobbs

The role of protein synthesis during metabolic depression in the Australian desert frog Neobatrachus centralis.

Little is known about the role of energy consuming processes during metabolic depression. We have shown that aestivation in the Australian desert frog Neobatrachus centralis is accompanied by an in vivo metabolic depression of 77%. Using an in vitro liver slice preparation, we have measured an in vitro metabolic depression in liver of 55%, with a concomitant 67% decrease in the rate of protein synthesis. The decrease in protein synthesis accounts for 52% of the metabolic depression of the tissue, but only 4.9% of the metabolic depression of the whole animal. No in vitro metabolic depression or decrease in protein synthesis during aestivation was measured in muscle, but a decrease in the low rate of protein synthesis in muscle in vivo could not, in any case, account for more than 3% of the metabolic depression of the whole animal. The liver, although not a quantitatively important tissue in terms of metabolic depression in vivo, offers the opportunity to characterise the regulation of protein synthesis in a system in which metabolic depression is not confounded by changes in ambient temperature and PO2.

Over-expression of cytochrome P450 CYP6B7 mRNA and pyrethroid resistance in Australian populations of Helicoverpa armigera (Hübner)

Three cDNA clones for cytochrome P450s, CYP6B2, CYP6B6 and CYP6B7 which have 84-87% protein sequence identity have been isolated previously from Helicoverpa armigera, and the CYP6B7 mRNA was found to be over-expressed in a pyrethroid-resistant field population. Subsequent analysis has shown that over-expression is observed in a majority of individuals in all populations tested. Single-pair crosses between resistant and sensitive individuals indicated that the pyrethroid resistance co-segregated with the over-expression of this mRNA. Southern analysis indicated that the over-expression, which was confined to midgut only in many insects, was not related to gene amplification. These observations add weight to the conclusion that CYP6B7 is the cytochrome P450 form involved in pyrethroid resistance, and that over-expression of cytochrome P450 CYP6B7 is a common cause of pyrethroid resistance in H. armigera. The results suggest that specific probes for CYP6B7 protein or mRNA could provide the basis for the development of tools for monitoring pyrethroid resistance due to mixed function oxidase activity in field populations of this insect.

The role of eukaryotic initiation factor 2 during the metabolic depression associated with estivation

SUMMARY We have investigated the role of eukaryotic initiation factor 2α (eIF2α) in two estivating organisms previously shown to downregulate protein synthesis during metabolic depression, the land snail Helix aspersa Müller and the desert frog Neobatrachus sutor Main 1957. We have developed a method using a single antibody (which binds specifically to the phosphorylated, conserved phosphorylation region) by which the total levels of eIF2α and the ratio of phosphorylated eIF2α [eIF2α(P)] to total (phosphorylated and unphosphorylated) eIF2α can be determined. In H. aspersa, we have shown that the level of eIF2α mRNA expression is unchanged between the awake and estivating states. The amount of total eIF2α is the same in the estivating and awake states, and eIF2α(P) is undetectable and must represent ≤10% of total eIF2α in both states. Conversely, in N. sutor during estivation, the level of total eIF2α increases approximately 1.6-fold and the ratio of eIF2α(P)/eIF2α increases from 0.22±0.11 to 0.52±0.08, implicating eIF2α phosphorylation in the downregulation of protein synthesis during estivation in this animal. The differences in the amounts of eIF2α and the level of its phosphorylation between these two species also suggest possible differences either in the mechanism by which protein synthesis is downregulated during estivation or in the sensitivity of the initiation of translation to eIF2α(P) levels.

Isolation and characterisation of a cytochrome b5 cDNA clone from Helicoverpa armigera (Hubner): possible involvement of cytochrome b5 in cytochrome P450 CYP6B7 activity towards pyrethroids

A cDNA clone specific for cytochrome b5 was isolated from Helicoverpa armigera. This sequence corresponded to a mRNA of an estimated 544 nucleotides in length excluding the poly A tail. The mRNA contained an open reading frame of 381 nucleotides encoding a protein of 127 amino acid residues with a molecular weight of 14,564 Daltons. The encoded protein sequence showed 51% protein sequence identity with cytochrome b5 from M. domestica and 36-37% identity with mammalian and avian cytochrome b5 sequences. Northern analysis of larval RNA using this cDNA as probe, revealed that cytochrome b5 mRNA expression is tissue specific with the mRNAs being expressed in abundance in the midguts of larvae, at a lower level in fatbody but is not detectable in larval integument. During normal development this mRNA was undetectable in eggs but was present at similar levels from first to fifth instar larvae. The mRNA was expressed at very low levels in pupae and adult moths. The cytochrome b5 mRNA was found to be inducible by treatment with the monoterpene, a-pinene, and to be over-expressed in some individuals of a pyrethroid resistant population of H. armigera. The induction and over-expression patterns were identical to the cytochrome P450, CYP6B7 mRNA. The present data suggests that cytochrome b5 may be involved in CYP6B7 mediated pyrethroid resistance in H. armigera.

A simple method to obtain the 5′ ends of mRNA sequences by direct ligation of cDNA–RNA hybrids to a plasmid vector

▼Partial cDNA clones from the 3′-ends of mRNAs can often be obtained using a degenerate primer to a conserved region of a protein in an anchored PCR reaction (Ref. 1). However, further sequencing requires some form of 5′RACE. The methods are PCR based and require the addition of a synthetic priming sequence to the 3′-end of the cDNA. This is usually done either using the enzyme, terminal deoxynucleotidyl transferase (Ref. 2, 3, 4), or by ligation of a singlestranded oligonucleotide to the 3′-end of the cDNA using RNA ligase (Ref. 5). However, we have found the terminal deoxynucleotidyl transferase enzyme very difficult to use and the RNA ligase procedure long and complicated and also unreliable. In this report we describe a simple technique to obtain 5′ends of mRNA by adding an artificial priming site to the 3′end of newly synthesized cDNA using T4 DNA ligase (Figure 1). The procedure relies on the fact that DNA–RNA hybrids can act as substrates for T4 DNA ligase (in fact such hybrid molecules can be ligated directly into a plasmid vector to create a library of cDNA clones, although the efficiency is quite low). In this procedure, newly synthesized cDNA is retained as a cDNA–RNA hybrid. This hybrid is then ligated, using T4 DNA ligase, to plasmid DNA digested with a restriction enzyme that leaves blunt ends. The plasmid DNA then supplies the external priming sequence for either a T7 primer or M13 sequencing primer. The main advantages of

Polysome profiles in awake and estivating snails (Otala lactea)

Abstract The land snail, Otala lactea , like many other organisms, depresses its metabolism in the face of environmental stress. Due to the extent of this depression, a decrease in the rate of protein synthesis is likely to be involved. In an attempt to quantitate changes in the rate of protein synthesis during metabolic depression in O. lactea we have determined polysome profiles from the hepatopancreas of awake and estivating snails. In both estivating and awake snails, the majority of ribosomes were present as monosomes and there was no evidence of qualitative changes with the state of the snail.