Identification and Characterization of a cDNA and the Structural Gene Encoding the Mouse Epithelial Membrane Protein-1

The PMP22/EMP/MP20 gene family includes four closely related proteins, peripheral myelin protein-22 (PMP22), epithelial membrane protein-1 (EMP-1), epithelial membrane protein-2 (EMP-2), and epithelial membrane protein-3 (EMP-3), which share amino acid identities ranging from 33 to 43%. In addition, the lens-specific membrane protein MP20 represents a more distant relative. Functionally, this family of proteins is likely to play important roles in the control of cell proliferation, cell differentiation, and cell death. In particular, mutations affecting thePMP22gene are responsible for various hereditary peripheral neuropathies in humans and mice. We report the isolation and characterization of a mouse EMP-1 cDNA and the correspondingemp-1gene. Mouse EMP-1 displays 93% amino acid identity to rat EMP-1 and 39% identity to mouse PMP22. The cDNA-predicted EMP-1 protein contains four putative membrane-associated domains and can beN-linked glycosylatedin vitro.EMP-1 is encoded by a single-copy gene with the positions of introns exactly conserved betweenemp-1andPMP22,corroborating the hypothesis that both genes belong to the same family. Computer-predicted structural domains of EMP-1 are partially mirrored by the exon/intron structure ofemp-1.Most interestingly, exon 4, which covers the potential second transmembrane domain, a small intracellular loop, and half of the third transmembrane domain, encodes the most highly conserved regions between the EMP-1 and PMP22 proteins and is also remarkably conserved in the MP20 gene, indicating some shared functional significance for this module in the PMP22/EMP/MP20 family.     

Molecular Cloning of the N-Terminus of GTBP

Defects in mismatch repair genes cause the genetic instability characteristic of hereditary nonpolyposis colorectal cancer and a subset of sporadic colon tumors. The newest member of the mismatch repair gene family,GTBP, has recently been identified as a partial cDNA. Here, we describe the isolation of its 5′ terminus, allowing definition of the entire coding region. Several polymorphisms within the 5′ end were identified and are presented.     

A novel family of linear plasmids with homology to plasmid pAL2-1 of Podospora anserina

Three recently isolated wild-type strains of the ascomycete Podospora anserina were analyzed for the presence of linear mitochondrial plasmids. In one of these strains, designated Wa6, at least 12 distinct plasmid-like elements were identified. From molecular analyses a minimum number of 78 individual linear molecules with proteins bound to their 5′ ends was estimated. In addition, the different members of this family of typical linear plasmids were shown to possess a common central region and terminal sequences which differ from one plasmid to another due to the presence of different numbers of a 2.4 kb sequence module. Finally, the pWa6 plasmids share a high degree of sequence similarity with pAL2-1, a linear plasmid previously identified in mitochondria of a long-lived mutant of P.anserina. A mechanism is proposed which explains the generation of these distinct, closely related extrachromosomal genetic traits.     

Comparative studies on the dynamics of crosslinked insulin

Molecular dynamics simulations were carried out on an insulin crosslinked between the N-terminal A chain and the C-terminal B chain to form a so-called mini-proinsulin: N -^A1-N -^B29-diaminosuberoyl insulin (DASI). To investigate the influence of crosslinking on the dynamics of the insulin moiety, the bridge was removed from a transient DASI structure and simulation was carried on independently with the then unlinked (ULKI) as well as with the crosslinked species. The effects of crystal packing and quaternary interactions were checked by simulating both types of monomers and dimers known from the hexamer structure. All simulations were compared to previous ones of native insulin. DASI shows general similarity to the native simulations in most parts of the structure. Deviations are visible in the segments to which the bridge is directly connected, i.e. their flexibility is reduced. Upon removal of the bridge the ULKI simulations reapproach those of native insulin. The influence of the bridge spreads over the whole molecule, but all of its main structural features remain intact. The simulations suggest that the displacement of the C-terminal B chain of native insulin, considered important for receptor interaction, is prevented by the bridge, which also partially shields some binding residues. This is in accordance with the poor biological potency of A1-B29-crosslinked insulins.Abbreviations DASI-insulin(DASI) bovineN -^A1-N -^B29-di-aminosuberoyl insulin - ULK-insulin (ULKI) Native beef insulin with the bridge of DASI removed     

Recruitment of plasma membrane voltage-dependent calcium-permeable channels in carrot cells.

Numerous biological assays and pharmacological studies have led to the suggestion that depolarization-activated plasma membrane Ca2+ channels play prominent roles in signal perception and transduction processes during growth and development of higher plants. The recent application of patch-clamp techniques to isolated carrot protoplasts has led to direct voltage-clamp evidence for the existence of Ca2+ channels activated by physiological depolarizations in the plasma membrane of higher plant cells. However, these voltage-dependent Ca2+ channels were not stable and their activities decreased following the establishment of whole-cell recordings. We show here that large pre-depolarizing pulses positive to 0 mV induced not only the recovery of Ca2+ channel activities, but also the activation of initially quiescent voltage-dependent Ca2+ channels in the plasma membrane (recruitment). This recruitment was dependent on the intensity and duration of membrane depolarizations, i.e. the higher and longer the pre-depolarization, the greater the recruitment. Pre-depolarizing pulses to +118 mV during 30 s increased the initial calcium currents 5- to 10-fold. The recruited channels were permeable to Ba2+ and Sr2+ ions. The data suggested that voltage-dependent Ca(2+)-permeable channels are regulated by biological mechanisms which might be induced by large pre-depolarizations of the plasma membrane. In addition, this study provides evidence for the existence in the plasma membrane of higher plant cells of a large number of voltage-dependent Ca2+ channels of which a major part are inactive and quiescent. It is suggested that quiescent Ca2+ channels can be rapidly recruited for Ca(2+)-dependent signal transduction.     

src-specific immunity in inbred chickens bearing v-src DNA- and RSV-induced tumors

Serological data identify a single major histocompatibility complex (MHC) class I locus in cattle. Molecular data, however, demonstrate the presence of at least two cattle MHC (BoLA) class I loci. To investigate the number of transcribed BoLA class I genes, we amplified cattle cDNA by using a single MHC class I-specific primer that hybridized to a conserved region of exon 4 and a non-specific 3 primer. Six BoLA class I cDNAs have been cloned and sequenced from a Bos taurus bull heterozygous for BoLA class I serological antigens, demonstrating the presence of a minimum of three loci. Sequence comparisons suggested that one of these cDNAs may be an unexpressed allele or the product of a nonclassical locus.The nucleotide sequence data reported in this paper have been submitted to the GenBank nucleotide sequence database and have been assigned the accession numbers U01186 and U01187.     

Involvement of protein phosphorylation in the sensitivity of photosystem II to strong illumination

Four types of differently phosphorylated hylakoids isolated from field grown spinach ( Spinacia oleracea L.) were tested for the sensitivity of photosystem II (PSII) to photoinactivation. Phosphorylation of light-harvesting II complexes (LHCII) protected PSII electron transfer from photoinhibitory damage, while the phosphorylation of the PSII core polypeptides slightly accelerated the decline of electron transfer during high irradiance treatment. Dephosphorylation of the CP43 apoprotein and PsbH protein by an alkaline phosphatase resulted in an extreme sensitivity of the thylakoids to strong illumination. The PSII photoinactivation of thylakoids with the impaired oxygen-evolving complex was found to be independent of phosphorylation.
The thylakoids of the thermophilic cyanobacterium Synechococcus elongates were used in order to compare the plants with an organism where LHCII complexes are missing and the PSII core proteins are not phosphorylated.     

Anion Selectivity of Slow Anion Channels in the Plasma Membrane of Guard Cells (Large Nitrate Permeability)

Closing of stomatal pores in the leaf epidermis of higher plants is mediated by long-term release of potassium and the anions chloride and malate from guard cells and by parallel metabolism of malate. Previous studies have shown that slowly activating anion channels in the plasma membrane of guard cells can provide a major pathway for anion efflux while also controlling K+ efflux during stomatal closing: Anion efflux produces depolarization of the guard cell plasma membrane that drives K+ efflux required for stomatal closing. The patch-clamp technique was applied to Vicia faba guard cells to determine the permeability of physiologically significant anions and halides through slow anion channels to assess the contribution of these anion channels to anion efflux during stomatal closing. Permeability ratio measurements showed that all tested anions were permeable with the selectivity sequence relative to Cl- of NO3- > Br- > F- ~ Cl- ~ I- > malate. Large malate concentrations in the cytosol (150 mM) produced a slow down-regulation of slow anion channel currents. Single anion channel currents were recorded that correlated with whole-cell anion currents. Single slow anion channels confirmed the large permeability ratio for nitrate over chloride ions. Furthermore, single-channel studies support previous indications of multiple conductance states of slow anion channels, suggesting cooperativity among anion channels. Anion conductances showed that slow anion channels can mediate physiological rates of Cl- and initial malate efflux required for mediation of stomatal closure. The large NO3- permeability as well as the significant permeabilities of all anions tested indicates that slow anion channels do not discriminate strongly among anions. Furthermore, these data suggest that slow anion channels can provide an efficient pathway for efflux of physiologically important anions from guard cells and possibly also from other higher plant cells that express slow anion channels.