Characterization of two allelic variants of a human pregnancy-specific glycoprotein gene.
The pregnancy-specific glycoproteins (PSGs) of the human placenta are a group of proteins that together with the carcinoembryonic antigens comprise a subfamily within the immunoglobulin superfamily. To study the control of PSG expression, we isolated and characterized PSG genes and identified cis-acting DNA elements in the 5'-flanking gene regions essential for PSG expression. Two overlapping PSG cosmid clones, which contain two allelic variants of a PSG gene (PSG12 and PSG12 psi), were isolated from an unamplified library made from a single individual. Cosmid 1 contains exons 1 (5'/L) and 2 (L/N) of the PSG12 gene located downstream of a previously identified PSG1-I gene. Cosmid 6 contains a portion of the PSG1-I gene lacking exons 1 and 2 upstream of a complete PSG12 psi transcription unit. Sequence comparison indicates that exons 5'/L and L/N in PSG12 and PSG12 psi are 99% identical, except that the L/N exon in the PSG12 psi gene contains a stop codon. Both PSG12 and PSG12 psi transcripts were detected in the human placenta, indicating that both genes are actively transcribed. However, the PSG12 psi gene may represent an allelic pseudogene variant of the PSG12 gene, because all identified PSGs contain a functional N-domain. Primer extension analysis showed that the PSG12 gene starts at a cluster of sites located at -106 to -104 base pairs with respect to the translation start site. In transient transfection assays using a chloramphenicol acetyltransferase reporter gene, we demonstrated that the -835 to -34 DNA region upstream of the translation start site of PSG12 or PSG12 psi contained both positive and negative elements that control PSG expression. Deletion analysis showed that nucleotides -172 to -34 in the PSG12 gene could function as a promoter. Gel retardation analysis showed that protein factors in human placental cell extract formed four complexes (I, II, IIa, and III) with the PSG12(-172/-34) DNA. Site-directed mutagenesis that prevents protein factor binding to the PSG12 promoter resulted in a marked reduction in transcription activation, locating the core enhancers at nucleotides -148 to -141 and -60 to -55. Mutagenesis studies also showed that the ACAGC repeats at nucleotides -84 to -68 in the PSG12 5'-flanking are essential for expression of the PSG12 gene in human placental cells.
Lei
K J
KJ
Human Genetics Branch, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland 20892.
Wang
C
C
Chamberlin
M E
ME
Liu
J L
JL
Pan
C J
CJ
Chou
J Y
JY
D000483:Alleles; D000595:Amino Acid Sequence; D001483:Base Sequence; D004247:DNA; D014644:Genetic Variation; D006023:Glycoproteins / Q000096:biosynthesis / Q000235:genetics*; D006801:Humans; D008969:Molecular Sequence Data; D010920:Placenta / Q000378:metabolism; D011257:Pregnancy Proteins / Q000096:biosynthesis / Q000235:genetics*; D011268:Pregnancy-Specific beta 1-Glycoproteins; D011401:Promoter Regions, Genetic; D011544:Pseudogenes; D012315:RNA Caps; D015183:Restriction Mapping; D012689:Sequence Homology, Nucleic Acid; D014158:Transcription, Genetic
D006023:Glycoproteins; D011257:Pregnancy Proteins; D011268:Pregnancy-Specific beta 1-Glycoproteins; D012315:RNA Caps; C082668:pregnancy-specific beta-1-glycoprotein 12; D004247:DNA