The mouse lens fiber-cell intrinsic membrane protein MP19 gene (Lim2) and granule membrane protein GMP-17 gene (Nkg7): Isolation and sequence analysis of two neighboring genes.
OBJECTIVE
The lens fiber cell intrinsic membrane protein MP19 appears to play a key role in lens fiber cell structure or communication, and thus cataractogenesis. The goal of this study was to isolate and characterize the entire gene structure of the MP19 gene, termed Lim2, and to investigate gene sequences surrounding this lens-specific gene.


METHODS
A 129/SvJ mouse genomic DNA library was screened using radioisotope labeled bovine MP19 cDNA. From this screening, an 11 kb genomic fragment was isolated which contained the entire Lim2 gene, and a neighboring gene, Nkg7, which codes for a 17 kDa granulocyte membrane protein termed GMP-17. The nucleotide sequence of this entire fragment was obtained using double strand automated sequencing techniques. Using CAT and green fluorescent protein reporter constructs, Lim2 5'-upstream promoter sequences were analyzed.


RESULTS
An 11,182 base pair genomic clone containing the entire murine Lim2 gene and another downstream gene, Nkg7, was obtained and completely sequenced. These two genes are only 1,182 base pairs apart, from the poly(A) signal of the Lim2 gene to the published transcriptional start site of Nkg7. Interestingly, the protein coded for by Nkg7, GMP-17, is very similar to the product of the lens Lim2 gene, MP19, in many respects. Both proteins are transmembrane proteins, with each having 4 transmembrane loops. The amino acid sequence of the two proteins is 34% identical, and 49% with respect to similar amino acids. The size of mouse Lim2 is 5,896 base pairs from the transcriptional start site to the poly(A) signal, and contains five exons and four introns. Exons 2-5 of the Lim2 gene encode a polypeptide of 173 amino acids, having over 92% identity to human MP19. Using chloramphenicol acetyltransferase (CAT) and green fluorescent protein (GFP) reporter constructs, it was determined that about 160 bp of sequence upstream from the start of transcription is both necessary and sufficient for efficient expression levels as well as tissue specificity of expression.


CONCLUSIONS
The mouse Lim2 gene is very similar to the human LIM2 gene, both having the same number of exons and introns. The coding nucleotide sequences from both species are 88% identical, and 92% identical at the amino acid level. In the immediate 5'-upstream region of these two genes, several highly conserved regions are observed. Due to the similarity of the MP19 and GMP-17 proteins, it is interesting to speculate that the lens MP19 and the lymphocyte-associated GMP-17 may have originated from one primordial gene which, through genetic drift, resulted in two separate proteins having similar functions in two widely separated tissue types.
Zhou
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Emory Eye Center, Department of Ophthalmology, Emory University School of Medicine, Atlanta, GA 30322, USA.
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Church
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D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002478:Cells, Cultured; D002642:Chick Embryo; D004847:Epithelial Cells / Q000378:metabolism; D005136:Eye Proteins / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D005347:Fibroblasts / Q000378:metabolism; D015870:Gene Expression; D015723:Gene Library; D049452:Green Fluorescent Proteins; D006801:Humans; D007908:Lens, Crystalline / Q000737:chemistry* / Q000196:embryology / Q000378:metabolism; D008164:Luminescent Proteins / Q000378:metabolism; D008562:Membrane Glycoproteins; D008565:Membrane Proteins / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D051379:Mice; D008969:Molecular Sequence Data; D039102:Poly(A)-Binding Proteins; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic / Q000235:genetics; D011506:Proteins; D016601:RNA-Binding Proteins / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D017422:Sequence Analysis, DNA; D000074019:T-Cell Intracellular Antigen-1; D014162:Transfection
D005136:Eye Proteins; C044539:LIM2 protein, human; C489025:Lim2 protein, mouse; D008164:Luminescent Proteins; D008562:Membrane Glycoproteins; D008565:Membrane Proteins; C499363:NKG7 protein, human; C499365:Nkg7 protein, mouse; D039102:Poly(A)-Binding Proteins; D011506:Proteins; D016601:RNA-Binding Proteins; D000074019:T-Cell Intracellular Antigen-1; C071558:TIA1 protein, human; D049452:Green Fluorescent Proteins