Major Intrinsic Protein Gene Research Articles

Functional characterization of a microbial aquaglyceroporin.

The major intrinsic proteins (MIPs) constitute a widespread membrane channel family essential for osmotic cell equilibrium. The MIPs can be classified into three functional subgroups: aquaporins, glycerol facilitators and aquaglyceroporins. Bacterial MIP genes have been identified in archaea as well as in Gram-positive and Gram-negative eubacteria. However, with the exception of Escherichia coli, most bacterial MIPs have been analysed by sequence homology. Since no MIP has yet been functionally characterized in Gram-positive bacteria, we have studied one of these members from Lactococcus lactis. This MIP is shown to be permeable to glycerol, like E. coli GlpF, and to water, like E. coli AqpZ. This is the first characterization of a microbial MIP that has a mixed function. This result provides important insights to reconstruct the evolutionary history of the MIP family and to elucidate the molecular pathway of water and other solutes in these channels.

INVITED EDITORIAL The Genetics of Cataract: Our Vision Becomes Clearer

I would like to thank Drs. Joe Horwitz, Muriel Kaiser-Kupfer, Mark Kantorow, Eric Wawrousek, and Sam Zigler for a close reading of the manuscript.

The major intrinsic protein family of Arabidopsis has 23 members that form three distinct groups with functional aquaporins in each group.

Aquaporins, proteins that enhance the permeability of biological membranes to water, are widely distributed in living organisms. They are 26- to 29-kD proteins that belong to the major intrinsic protein (MIP) family of channels. By searching the Arabidopsis thaliana expressed sequence tag database and by using the polymerase chain reaction with oligonucleotides to conserved plant aquaporin domains, we identified 23 expressed Arabidopsis MIP genes. Eight of these had been previously identified as active aquaporins, and two additional ones are now reported to have water-transport activity in Xenopus laevis oocytes. One of these is highly expressed in suspension-cultured cells. On a dendrogram these 23 MIP sequences cluster into three groups: the first group has 11 members and contains the plasma membrane aquaporins, the second group also has 11 members and contains the tonoplast aquaporins, and the third group has only a single member. This MIP protein, provisionally called At-NLM1, is most closely related to the Gm-NOD26 protein that is found in the bacteroid membranes of soybean (Glycine max L.) nodules; At-NLM1 is an active aquaporin when expressed in oocytes. With a semiquantitative slot-blot analysis technique, we determined the expression levels of 22 MIP genes in the various organs. The quantitative polymerase chain reaction was used to determine the effects of various stress treatments on the expression of NLM1.

Mutations in the founder of the MIP gene family underlie cataract development in the mouse.

The major intrinsic protein (MIP) of the vertebrate eye lens is the first identified member of a sequence-related family of cell-membrane proteins that appears to have evolved by gene duplication. Several members of the MIP family transport water (aquaporins), glycerol and other small molecules in microbial, plant and animal cells. Mutations in two aquaporin homologues of MIP underlie an autosomal recessive form of nephrogenic diabetes insipidus and absence of the Colton blood group antigens in humans, whereas, mutation of a third MIP-like gene underlies 'big brain' development in Drosophila. Here we show that distinct mutations in the murine Mip gene underlie one form of autosomal dominant cataract in the mouse. The cataract Fraser mutation is a transposon-induced splicing error that substitutes a long terminal repeat sequence for the carboxy-terminus of MIP. The lens opacity mutation is an amino-acid substitution that inhibits targeting of MIP to the cell-membrane. These allelic cataract mutations provide the first direct evidence that MIP plays a crucial role in the development of a transparent eye lens.

Isolation and characterization of the 5′-flanking sequence of the human ocular lens MIP gene

The MIP (major intrinsic protein) gene, a member of an ancient family of membrane channel genes, encodes the predominant fiber cell membrane protein of the ocular lens. Its specific expression in the lens fibers is temporally and spatially regulated during development. To study the regulation of expression of MIP and delineate the regulatory elements underlying its tissue specificity and ontogenic profile, we have cloned 2840 bp of the human MIP 5′-flanking sequence. The human MIP 5′-flanking sequence contains three complete Alu repetitive elements in tandem at position between nt −1699 and −2684 (nt −1699/−2684). These Alu elements appear to have had a complex evolutionary history with insertions at different times. We have fused DNA fragments containing MIP 5′-flanking sequences to the bacterial cat reporter gene encoding chloramphenicol acetyltransferase and assayed them in primary cultures of chicken lens cells. We have mapped two negative regulatory regions in the human MIP 5′-flanking sequences −1564/−1696 and −948/−1000. We demonstrated that the human MIP 5′-flanking sequence −253/+42 contains a functional promoter in lens cells but is inactive in kidney epithelial cells or mouse fibroblasts, suggesting that this sequence contains regulatory elements responsible for the lens-specific expression of MIP.

Human AQP2 and MIP genes, two members of the MIP family, map within chromosome band 12q13 on the basis of two-color FISH

The human AQP2 (collecting duct water channel, aquaporin 2) gene encodes a 271 amino acid protein and is a member of the MIP (major intrinsic protein of lens fiber) gene family. Using two-color fluorescence in situ hybridization on high-resolution R-banded chromosomes and human genomic DNA clones for AQP2 and MIP as probes, we found that both genes mapped closely within the human chromosome region 12q13.

Isolation of human aquaporin-CD gene.

The human gene encoding aquaporin-CD (AQP-CD) was isolated, and its structural organization was characterized. The gene appeared to exist as a single copy in the human genome and comprises four exons distributing over 5 kilobases. The size range of exons is 81-761 base pairs, and that for introns is approximately 3000 to approximately 250 base pairs. The exon-intron boundaries of human AQP-CD gene are identified at identical positions in other related genes, the human AQP-CHIP gene and the human major intrinsic protein gene. The major transcription initiation sites were identified to positions 93 and 94 base pairs upstream of the ATG initiation codon by primer extension and ribonuclease protection assay. The 5'-flanking region of the hAQP-CD gene was characterized by a TATA box, two GATA consensus sequences, an AP-1 site, an AP-2 site, three E-boxes, and a cyclic AMP-responsive element. These structural features will lead to a better understanding of the mechanisms of tissue-specific expression and the regulation by dehydration in AQP-CD gene and will also be of help in search for possible genetic disorders in human AQP-CD gene.

In situ hybridisation localises the gene for the major intrinsic protein of eye lens fibre cell membranes to human chromosome 12q14.

The gene encoding the major intrinsic protein (MIP) of eye lens fibre cell membranes has been localised to human chromosome 12q14 by in situ hybridisation of a cDNA for rat MIP to G-banded metaphase chromosomes. The human MIP gene maps within a conserved region of synteny with mouse Chromosome 10.

Genomic cloning, complete nucleotide sequence, and structure of the human gene encoding the major intrinsic protein (MIP) of the lens

Major intrinsic protein (MIP, also called MP26) is the predominant fiber cell membrane protein of the ocular lens. MIP has been suggested to play a role in cell-cell communication in the lens. Its expression is tissue-specific and developmentally regulated. We have isolated and characterized the human gene encoding MIP and report here its genomic structure and entire nucleotide sequence. The gene is 3.6 kb, contains four exons separated by introns ranging in size from 0.4 to 1.6 kb, and is present in single copy per haploid human genome. Primer extension of human lens RNA indicates that transcription of the gene initiates from a single site 26 nt downstream from the TATA box. Three complete Alu repetitive elements are found in tandem in the 5′-flanking region of the gene, and a single complete Alu sequence is present in the third intron. The interspecies comparisons of the MIP gene coding sequence and homologies to other members of a putative transmembrane channel protein superfamily are also discussed.