Localisation of the gene for the major intrinsic protein of eye-lens-fibre cell membranes to mouse chromosome 10 by in situ hybridisation.

The gene encoding the major intrinsic protein (Mip) of eye-lens-fibre cell membranes has been assigned to region D1 of mouse Chromosome 10 by in situ hybridisation of a cDNA for rat MIP to G-banded metaphase chromosomes. The mouse Mip gene maps within or near to a segment homoeologous with human chromosome 12q and may be linked to the Cat locus at the distal end of mouse Chromosome 10.     

Restriction fragment length polymorphisms associated with the gene for the major intrinsic protein of eye-lens fibre cell membranes in mice with hereditary cataracts

Cloned cDNAs coding for eye-lens fibre cell-membrane proteins, MIP and MP70, were used to detect restriction fragment length polymorphisms (RFLPs) in genomic DNA from inbred mice with autosomally inherited cataracts. Whereas distinct RFLPs associated with the MIP gene were identified in the Cba Cat and Nct mutants, no such genetic variation was associated with the MP70 gene. RFLPs associated with the mouse MIP gene may provide informative DNA markers in gene linkage studies of murine hereditary cataracts.     

Restriction fragment length polymorphisms associated with the gene for the major intrinsic protein of eye-lens fibre cell membranes in mice with hereditary cataracts.

Cloned cDNAs coding for eye-lens fibre cell-membrane proteins, MIP and MP70, were used to detect restriction fragment length polymorphisms (RFLPs) in genomic DNA from inbred mice with autosomally inherited cataracts. Whereas distinct RFLPs associated with the MIP gene were identified in the Cba Cat and Nct mutants, no such genetic variation was associated with the MP70 gene. RFLPs associated with the mouse MIP gene may provide informative DNA markers in gene linkage studies of murine hereditary cataracts.     

Constitutive expression of a Mg2+-inhibited K+ current and a TRPM7-like current in human erythroleukemia cells

Whole cell patch-clamp experiments were undertaken to define the basal K(+) conductance(s) in human erythroleukemia cells and its contribution to the setting of resting membrane potential. Experiments revealed a non-voltage-activated, noninactivating K(+) current. The magnitude of the current recorded under whole cell conditions was inhibited by an increase in free intracellular Mg(2+) concentration. Activation or inactivation of the Mg(2+)-inhibited K(+) current (MIP) was paralleled by activation or inactivation of a Mg(2+)-inhibited TRPM7-like current displaying characteristics indistinguishable from those reported for molecularly identified TRPM7 current. The MIP and TRPM7 currents were inhibited by 5-lipoxygenase inhibitors. However, inhibition of the MIP current was temporally distinct from inhibition of TRPM7 current, allowing for isolation of the MIP current. Isolation of the MIP conductance revealed a current reversing near the K(+) equilibrium potential, indicative of a highly K(+)-selective conductance. Consistent with this finding, coactivation of the nonselective cation current TRPM7 and the MIP current following dialysis with nominally Mg(2+)-free pipette solution resulted in hyperpolarized whole cell reversal potentials, consistent with an important role for the MIP current in the setting of a negative resting membrane potential. The MIP and TRPM7-like conductances were constitutively expressed under in vivo conditions of intracellular Mg(2+), as judged by their initial detection and subsequent inactivation following dialysis with a pipette solution containing 5 mM free Mg(2+). The MIP current was blocked in a voltage-dependent fashion by extracellular Cs(+) and, to a lesser degree, by Ba(2+) and was blocked by extracellular La(3+) and 2-aminoethoxydiphenyl borate. MIP currents were unaffected by blockers of ATP-sensitive K(+) channels, human ether-à-go-go-related gene current, and intermediate-conductance Ca(2+)-activated K(+) channels. In addition, the MIP current displayed characteristics distinct from conventional inwardly rectifying K(+) channels. A similar current was detected in the leukemic cell line CHRF-288-11, consistent with this current being more generally expressed in cells of leukemic origin.     

Evolution of the gene encoding mitochondrial intermediate peptidase and its cosegregation with the A mating-type locus of mushroom fungi

The high level of DNA polymorphism at the mating-type loci of mushroom fungi has made the cloning of mating-type genes difficult. As an alternative to strategies that employ sequence conservation, an approach utilizing conserved gene order could facilitate the cloning of A mating-type genes from mushroom fungi. It has been shown that a gene encoding a mitochondrial intermediate peptidase (MIP) is very close ( < 1 kbp) to the A mating-type locus of two model mushroom species. In this study, the cosegregation of MIP and the A mating-type locus was studied by genotyping progeny of seven additional mushroom species using PCR and genetic crosses. No evidence of any recombination between MIP and the A mating-type locus was detected among all seven species. Phylogenetic analysis of MIP sequences from diverse mushroom species agrees with the current organismal phylogeny, suggesting the sequences are generally orthologous.     

Isolation of the Ascobolus Immersus Spore Color Gene B2 and Study in Single Cells of Gene Silencing by Methylation Induced Premeiotically

The ascomycete Ascobolus immersus has been extensively used as a model system for the genetic study of meiotic recombination. More recently, an epigenetic process, known as methylation induced premeiotically (MIP), that acts on duplicated sequences has been discovered in A. immersus and has raised a new interest in this fungus. To try and extend these studies, we have now cloned the A. immersus spore color gene b2, a well characterized recombination hot-spot. Isolation of the whole gene was verified by physical mapping of four large b2 alterations, followed by transformation and mutant rescue of a null b2 allele. Transformation was also used to duplicate b2 and subject it to MIP. As a result, we were able for the first time to observe gene silencing as early as just after meiosis and in single cells. Furthermore, we have found evidence for a modulating effect of MIP on b2 expression, depending on the region of the gene that is duplicated and hence subjected to MIP.     

The major intrinsic protein (MIP) of the bovine lens fiber membrane: characterization and structure based on cDNA cloning

Synthetic oligonucleotide probes have been used to identify two overlapping cDNA clones that represent the entire coding region of the mRNA for the major intrinsic protein (MIP) of bovine lens cell membrane. Hybridization studies indicate that bovine MIP is encoded by a single-copy gene. The cDNA hybridizes to the rat genome, but MIP mRNA is not detected in rat liver. Analysis of the deduced amino acid sequence provides support for the potential role of MIP as a junctional protein. The structure predicted for MIP suggests that it traverses the lipid bilayer six times with both carboxy and amino termini on the cytoplasmic side, and that it has at least one amphiphilic transmembrane segment, as expected if the protein were to participate in the formation of an aqueous channel.     

Sodium butyrate suppresses the transforming activity of an activated N-ras oncogene in human colon carcinoma cells

The transforming activity of DNA from a newly established undifferentiated human colon carcinoma cell line (MIP-101) was tested in the NIH-3T3 transfection assay. Southern blot analysis of the transfectant DNA revealed the presence of a human N-ras oncogene. Treatment of MIP-101 cells with the maturational agent sodium butyrate induced a more normal phenotype, including diminished growth rate, elimination of anchorage independent growth, and decreased tumorigenicity (R. Niles, S. Wilhelm, P. Thomas, and N. Zamcheck (1988) J. Cancer Invest. 6, 39). Here we report that there is a significant reduction in the transforming efficiency of the DNA from butyrate-treated MIP-101 cells. A nonspecific reduction in total DNA uptake as an explanation for these findings was eliminated by showing that there was similar uptake and expression of the thymidine kinase gene from the DNA of butyrate-treated and control MIP cells. Butyrate treatment had no detectable effect on the overall structure, methylation, and level of expression of the human N-ras gene from MIP-101 cells. An NIH-3T3 transformant ability after treatment with sodium butyrate. Although butyrate suppressed several transformed properties similar to MIP-101 cells, DNA from control and treated cultures had an identical level of transforming activity. The results suggest that the environment of the MIP cells may contain additional elements not present in the NIH-3T3 transformants which are required to observe the effect of butyrate on reduction of transforming activity.     

Covalent change of major intrinsic polypeptide (MIP26K) of lens membrane during human senile cataractogenesis

Polyclonal antisera have been made against synthetic peptides corresponding to the C-terminal octapeptide and N-terminal nonapeptide of bovine MIP26K. Western blot analysis demonstrated significant binding of the C-terminal antiserum to MIP26K of both normal and cataractous human lens. In contrast, the N-terminal antiserum bound to MIP26K of normal lenses, but failed to bind to MIP26K of 7 out of 10 cataractous lenses studied. These results demonstrate for the first time, a covalent change in MIP26K during human cataractogenesis, and strongly suggest that the location of this change is in the N-terminal region of the polypeptide.     

Predicting Positive p53 Cancer Rescue Regions Using Most Informative Positive (MIP) Active Learning

Many protein engineering problems involve finding mutations that produce proteins with a particular function. Computational active learning is an attractive approach to discover desired biological activities. Traditional active learning techniques have been optimized to iteratively improve classifier accuracy, not to quickly discover biologically significant results. We report here a novel active learning technique, Most Informative Positive (MIP), which is tailored to biological problems because it seeks novel and informative positive results. MIP active learning differs from traditional active learning methods in two ways: (1) it preferentially seeks Positive (functionally active) examples; and (2) it may be effectively extended to select gene regions suitable for high throughput combinatorial mutagenesis. We applied MIP to discover mutations in the tumor suppressor protein p53 that reactivate mutated p53 found in human cancers. This is an important biomedical goal because p53 mutants have been implicated in half of all human cancers, and restoring active p53 in tumors leads to tumor regression. MIP found Positive (cancer rescue) p53 mutants in silico using 33% fewer experiments than traditional non-MIP active learning, with only a minor decrease in classifier accuracy. Applying MIP to in vivo experimentation yielded immediate Positive results. Ten different p53 mutations found in human cancers were paired in silico with all possible single amino acid rescue mutations, from which MIP was used to select a Positive Region predicted to be enriched for p53 cancer rescue mutants. In vivo assays showed that the predicted Positive Region: (1) had significantly more (p<0.01) new strong cancer rescue mutants than control regions (Negative, and non-MIP active learning); (2) had slightly more new strong cancer rescue mutants than an Expert region selected for purely biological considerations; and (3) rescued for the first time the previously unrescuable p53 cancer mutant P152L.     

MIP1, a new yeast gene homologous to the rat mitochondrial intermediate peptidase gene, is required for oxidative metabolism in Saccharomyces cerevisiae.

Cleavage of amino-terminal octapeptides, F/L/IXXS/T/GXXXX, by mitochondrial intermediate peptidase (MIP) is typical of many mitochondrial precursor proteins imported to the matrix and the inner membrane. We previously described the molecular characterization of rat liver MIP (RMIP) and indicated a putative homolog in the sequence predicted from gene YCL57w of yeast chromosome III. A new yeast gene, MIP1, has now been isolated by screening a Saccharomyces cerevisiae genomic library with an RMIP cDNA probe. MIP1 predicts a protein of 772 amino acids (YMIP), which is 54% similar and 31% identical to RMIP and includes a putative 37-residue mitochondrial leader peptide. RMIP and YMIP contain the sequence LFHEMGHAM HSMLGRT, which includes a zinc-binding motif, HEXXH, while the predicted YCL57w protein contains a comparable sequence with a lower degree of homology. No obvious biochemical phenotype was observed in a chromosomally disrupted ycl57w mutant. In contrast, a mip1 mutant was unable to grow on nonfermentable substrates, while a mip1 ycl57w double disruption did not result in a more severe phenotype. The mip1 mutant exhibited defects of complexes III and IV of the respiratory chain, caused by failure to carry out the second MIP-catalyzed cleavage of the nuclear-encoded precursors for cytochrome oxidase subunit IV (CoxIV) and the iron-sulfur protein (Fe-S) of the bc1 complex to mature proteins. In vivo, intermediate-size CoxIV was accumulated in the mitochondrial matrix, while intermediate-size Fe-S was targeted to the inner membrane. Moreover, mip1 mitochondrial fractions failed to carry out maturation of the human ornithine transcarbamylase intermediate (iOTC), specifically cleaved by RMIP. A CEN plasmid-encoded YMIP protein restored normal MIP activity along with respiratory competence. Thus, YMIP is a functional homolog of RMIP and represents a new component of the yeast mitochondrial import machinery.     

Amino-terminal octapeptides function as recognition signals for the mitochondrial intermediate peptidase.

We have shown previously that cleavage of a number of precursors by the mitochondrial processing peptidase (MPP) requires an intermediate octapeptide (FXXSXXXX) between the MPP cleavage site and the mature protein amino terminus. We show now that these octapeptides, present at the amino termini of the intermediates, direct recognition of these substrates by the mitochondrial intermediate peptidase (MIP), leading to formation of mature proteins. Synthetic peptides, corresponding to the intermediate octapeptides of human ornithine transcarbamylase (OTC) and of Neurospora cytochrome c reductase Fe/S subunit (Fe/S), inhibit the processing activity of purified rat liver MIP in vitro, without affecting MPP activity; this indicates that the octapeptides can be recognized by MIP independent of the presence of the corresponding mature proteins and interact with a site that is crucial for MIP activity. MIP activity is not inhibited by a peptide lacking the amino-terminal hydrophobic residue, while substitution of such a residue by a polar amino acid causes a 10-fold reduction in the efficiency of MIP inhibition. To analyze the requirements for removal of the octapeptide from the intermediate proteins by MIP, artificial intermediates were synthesized and subjected to in vitro processing by purified MIP. The octapeptide can be cleaved by MIP only when the amino-terminal hydrophobic residue is also the amino terminus of the intermediate. Further, when the OTC octapeptide is joined to the mature amino terminus of another twice-cleaved precursor (pFe/S; rat malate dehydrogenase, pMDH), the chimeric intermediate is cleaved by MIP to the corresponding mature-sized protein. When the OTC octapeptide is joined to the mature amino terminus of a once-cleaved precursor (yeast F1-beta-ATPase, pF1-beta), however, this intermediate is not cleaved by MIP; rather, it is processed by MPP to mature-sized F1-beta. Therefore, amino-terminal octapeptides can be cleaved by MIP only within the structural context of twice-cleaved precursors.     

Identification and functional analysis of the MOC1 interacting protein 1

Rice tillering is one of the most important agronomic traits that determine grain yields.Our previous study has demonstrated that the MONOCULM1(MOC1)gene is a key component that controls the formation of rice tiller buds.To further elucidate the molecular mechanism of MOC1 involved in the regulation of rice tillering.we performed a yeast-two-hybrid screening to identify MOC1 interacting proteins(MIPs).Here we reported that MIP1 interacted with MOC1 both in vitro and in vivo.The overexpression of MIP1 resulted in enhanced tillering and reduced plant height.In-depth characterization of the context of MIP1 and MOC1 would further our understanding of molecular regulatory mechanisms of rice tillering.     

Thin Filament Proteins and Thin Filament-Linked Regulation of Vertebrate Muscle Contractio

Abstract

The major intrinsic protein (MIP) of the bovine lens fiber cell membrane was the first member of the MIP family of proteins to be sequenced and characterized. It is probably a homotetramer with transmembrane channel activity that plays a role in lens biogenesis or maintenance. The polypeptide chain of each subunit may span the membrane six times, and both the N- and C-termini face the cell cytoplasm. Eighteen sequenced or partially sequenced proteins from bacteria, yeast, plants, and animals have now been shown to be members of the MIP family. These proteins appear to function in (1) metazoan development and neurogenesis (MIP and BIB), (2) water transport across the human erythrocyte membrane (ChIP), (3) communication between host plant cells and symbiotic nitrogen-fixing bacteria (NOD), (4) transport across the tonoplast membrane during plant seed development (α-TIP), (5) water stress-induced resistance to desiccation in plants (Wsi-TIP), (6) suppression of a genetic growth defect on fermentable sugars in yeast (FPS1), and (7) transport of glycerol across bacterial cell membranes (GlpF). One other sequenced member of the MIP family (ORF1 of Lactococcus lactis) has no known physiological function. The biochemical functions of the eukaryotic proteins are not well established.

Computer analyses have revealed that the first and second halves of all MTP family proteins probably arose by a tandem, intragenic, duplication event. Thus, the primary structure of putative transmembrane helices 1 to 3 is similar to that of putative transmembrane helices 4 to 6 even though they are of opposite orientation in the membrane. Among the most conserved residues in these two repeated halves are a membrane-embedded glutamate (E) in helices 1 and 4, an asparagine-proline-alanine (NPA) sequence in the loops between helices 2 and 3 (cytoplasmically localized) and helices 5 and 6 (extracellularly localized), and a glycine within helices 3 and 6. Statistical analyses suggest that the two halves of these proteins have evolved to serve distinct functions: the first half is more important for the generalized or common functions of these proteins, while the second half of these proteins is more differentiated to provide specific or dissimilar functions of the proteins. The apparent origin of MIP family proteins by duplication of a three-spanner precursor protein suggests an evolutionary origin distinct from other transport proteins with six transmembrane spanners. Based on the phylogenetic tree for the 18 sequenced members of the MTP family, we propose that a single, primordial gene arose in prokaryotes shortly before the emergence of eukaryotes, mat this gene was vertically transmitted to the principal eukaryotic kingdoms, and that subsequent gene duplication and divergence events gave rise to kingdom-related subfamilies or clusters of the MIP family.