The genes of major lysosomal membrane glycoproteins, lamp-1 and lamp-2. 5'-flanking sequence of lamp-2 gene and comparison of exon organization in two genes.

R Sawada,K.A Jardine,M Fukuda

doi:10.1016/s0021-9258(18)52972-0

Abstract

Human lysosomal membrane glycoproteins lamp-1 and lamp-2 are the major sialoglycoproteins present in lysosomal membranes. The expression of lamp-2 molecules is uniquely regulated, whereas lamp-1 is constitutively synthesized. In order to investigate the unique expression of lamp-2, and the gene evolution of lamp-1 and lamp-2, we isolated genomic phage clones encoding these glycoproteins. Comparison of the genomic and cDNA sequences revealed that the lamp-2 gene consists of nine exons. The transcriptional start site of the lamp-2 gene was determined by primer extension analysis. In order to locate the transcriptional regulatory region of this gene, various regions of 5'-sequences were tested for promoter activity using chloramphenicol acetyltransferase as a reporter molecule. The results revealed that the 5'-flanking sequence from -172 to -20 base pairs has strong promoter activity. In this sequence, potential SP1 and AP-1 binding sites and CAAT boxes are found. Most notably, the promoter activity is suppressed if the 5' farther upstream KpnI repeat sequence is included in the tested 5'-flanking sequence, thus suggesting that the KpnI repeat sequence may have some regulatory function in the lamp-2 gene expression. Comparison of the exon organization of human lamp-2 and lamp-1 genes, or chicken lamp-1 gene, reveals that these two proteins utilize the same exon phase in corresponding introns. Furthermore, each exon encodes almost identical portions of the proteins. On the other hand, the amino acid sequence of human lamp-1 is more homologous to lamp-1 of other species than it is to human lamp-2. These results indicate that lamp-1 and lamp-2 genes were most likely produced by duplication of a primordial gene, which took place early in evolution.

Highlights

Human lysosomal membrane glycoproteins lamp-1 Among this class of lysosomal membrane glycoproteins are and lamp-2are the major sialoglycoproteins present in two members, lamp-1’ and lamp-2, which have similar struclysosomal membranes
In order to determine thegenomic organization,each genomicclone was digestedbyvarious restriction enzymes and the genomic DNAfragmentsproduced were screened with the specific cDNA fragments used for screening the genomic library
Those positive DNA fragments were subcloned into a Bluescript vector

Summary

RESULTS

Isolation and Characterization of Human Lamp-2 Genomic Clones-The placental genomic DNA library was screened, initially withwhole lamp-2 cDNA but eventually withcDNA fragments harboring different parts of the cDNA sequence. By using four different probes in separate screening experiments, genomic clones harboring exon I, exon I1 and exon111, exon IV-VIII, and exon IX, respectively, were isolated in a frequency of one, one, two, and two out of lo6plaques. In order to determine thegenomic organization,each genomicclone was digestedbyvarious restriction enzymes and the genomic DNAfragmentsproduced were screened with the specific cDNA fragments used for screening the genomic library. Aphysical map of each exon was determined, after partialdigestion of genomic clonesfollowed by Southern blot hybridization, using probes specific to each exon. The results obtainedby these experiments are summarized Fiings.

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DISCUSSION

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