Association of feather colour with constitutively active melanocortin 1 receptors in chicken.

Seven alleles of the chicken melanocortin (MC) 1 receptor were cloned into expression vectors, expressed in mammalian cells and pharmacologically characterized. Four of the clones e(+R), e(+B&D), e(wh)/e(y), E(Rfayoumi) gave receptors to which melanocortin stimulating hormone (alpha-MSH) and NDP-MSH bound with similar IC50 values and responded to alpha-MSH by increasing intracellular cAMP levels in a dose-dependent manner. Three of the cMC1 receptors; e(b), E and E(R), did not show any specific binding to the radioligand, but were found to be constitutively active in the cAMP assay. The E and E(R) alleles are associated with black feather colour in chicken while the eb allele gives rise to brownish pigmentation. The three constitutively active receptors share a mutation of Glu to Lys in position 92. This mutation was previously found in darkly pigmented sombre mice, but constitutively active MC receptors have not previously been shown in any nonmammalian species. We also inserted the Glu to Lys mutation in the human MC1 and MC4 receptors. In contrast with the chicken clones, the hMC1-E94K receptor bound to the ligand, but was still constitutively active independently of ligand concentration. The hMC4-E100K receptor did not bind to the MSH ligand and was not constitutively active. The results indicate that the structural requirements that allow the receptor to adapt an active conformation without binding to a ligand, as a consequence of this E/K mutation, are not conserved within the MC receptors. The results are discussed in relationship to feather colour in chicken, molecular receptor structures and evolution. We suggest that properties for the 'E92K switch' mechanism may have evolved in an ancestor common to chicken and mammals and were maintained over long time periods through evolutionary pressure, probably on closely linked structural features.     

Cloning genomic sequences using long-range PCR

Protocols are presented for preparing DNA from a genomic library in λ phage and for synthesizing genomic fragments using PCR with nested vector- and gene-specific primers and linker-primers. Library DNA, isolated fromE. coli liquid lysates by a simple protocol, is used as template in PCR following a commercial protocol. The method produces library DNA sufficient for several hundred PCRs, incorporates nested primers to reduce nonspecific product formation, and enables the synthesis of linker-containing DNA fragments containing selected restriction sites to simplify subsequent cloning. The isolation of 5′ upstream sequences of three different arabidopsis genes by this methodod is described.     

The enteropathogenic Escherichia coli type III secretion system effector Map binds EBP50/NHERF1: implication for cell signalling and diarrhoea

Enteropathogenic Escherichia coli (EPEC) is the single most important contributor to child diarrhoea in developing countries. Nevertheless, the mechanism responsible for EPEC diarrhoea remains elusive. Using the yeast two-hybrid system to determine the target host cell protein of the EPEC type III secretion system effector Map led to identification of ezrin/radixin/moesin (ERM)-binding phosphoprotein 50 (EBP50), also known as Na+/H+ exchanger regulatory factor 1 (NHERF1). Protein interaction is mediated by the carboxy-terminal Thr-Arg-Leu (TRL) motif of Map and the PSD-95/Disk-large/ZO-1 domain 1 (PDZ1) of EBP50/NHERF1. Although EBP50/NHERF1 is recruited to site of EPEC adhesion in a Map-independent mechanism, co-immunoprecipitation and immunostaining revealed that Map binds to, induces proteolysis of, and colocalizes with EBP50/NHERF1 during infection of cultured epithelial cells. The TRL motif of Map was involved in Map-induced filopodia formation and brush border elongation on infected HeLa and Caco-2 cells respectively. As EBP50/NHERF1 regulates ion channels in the intestine we assessed the involvement of Map in diarrhoea using the Citrobacter rodentium mouse model of EPEC. We report significantly greater diarrhoea following infections with wild-type C. rodentium compared with C. rodentiumDeltamap. These results provide new insights into the mechanisms of EPEC diarrhoea.     

Tubal ligation in the chimpanzee (Pan troglodytes) as a means of contraception

Contraception of two chimpanzees was considered necessary for clinical reasons. After failure of reversible methods of contraception (an intrauterine device and a long-acting progestagen), tubal ligation was successfully performed, using a technique similar to one used in women.     

Reference cDNA library facilities available from European sources

cDNA libraries are the cornerstone of efforts to identify the relatively small regions of genomes that are responsible for biological effects. Gene hunter seeking candidate genes, via a variety of approaches, ultimately focus on the cloning, sequencing, and expression of cDNAs. Assistance is now available to researchers in the form of genome programs, whose initial goals include assembly of a complete collection of expressed sequences derived from the genome of interest. The concept of reference sets of cDNA libraries is that the aims of genome programs are served most effectively by different laboratories working on a common set of high-quality arrayed cDNA libraries, using different experimental approaches, thereby reducing unnecessary duplication of effort, and maximizing the amount of information that one set of resources can provide.     

Locust haemolymph lipoproteins visualised in the electron microscope

Summary In matureLocusta, the haemolymph lipoproteins change both in quality and quantity according to the physiological state of the animal. In resting locusts the majority of lipid in the haemolymph is carried by lipoprotein A_yellow, but during flight or after adipokinetic hormone injection, A_yellow joins together with extra diacylglycerols from the fat body and non-lipid carrying C_L-proteins to form another lipoprotein, A⁺. In this study partially purified A_yellow and A⁺ lipoproteins have been visualised by transmission electron microscopy after negative staining or shadowing. Both A_yellow and A⁺ lipoproteins are discrete particulate structures but they differ markedly in size; A_yellow particles are 9–16 nm in diameter while those of A⁺ are mostly in the range 20–50 nm. Large lipoprotein particles of the A⁺ type have not been described previously in insect haemolymph but, interestingly, the locust A⁺ particles do resemble most closely the low density lipoprotein particles described in human serum by Forte and Nichols (1972).     

Reversion from Suppression to Nonsuppression in SUQ5 [psi+] Strains of Yeast: The Classification of Mutations

Reversion from the suppressed to nonsuppressed phenotype in strains of genotype SUQ5 [psi⁺] ade2-1 his5-2 lys1-1 can1-100 ura3-1 has been induced by treatment with ethyl methanesulphonate, nitrosoguanidine or UV (254 nm) light. Spontaneously occurring revertants have also been selected by two different methods. Reversion has been shown to occur through a variety of nuclear mutations and through mutation of [psi⁺] to [psi^-]. Nuclear mutations included back-mutation of SUQ5, antisuppressor mutations that were recessive, semi-dominant or dominant, and dominant or recessive mutations of genes required for the maintenance of the [psi⁺] factor. Complementation tests by which the various kinds of mutations could be distinguished from one another were designed. The spectra of spontaneously occurring and induced mutations have been described.     

Differential expression of two related organ-specific genes in pea

We have screened a pea genomic library using a cDNA probe derived from pea shoot RNA. From this screen, we isolated two closely related genes, designated as S2 and P4. An intriguing property of these two genes is the presence in their coding region of a repeated sequence that is conserved between them in sequence but not in the number of the repeating units. The predicted amino acid sequence suggests that these proteins could be exported and glycosylated. 3 S1 analysis reveals that one of the genes, S2, is expressed highly in stem, as expected from previous work. However, mRNA derived from the other gene, P4, is not detectable in stem tissue, but is present in tissue derived from pea pods. The 5 upstream sequence of S2 and P4 are 94% identical up to position -121, suggesting that sequences upstream of -121 are responsible for organ-specific expression of the two genes.