Immunochemical, genetic and morphological comparison of fucosylation mutants of Dictyostelium discoideum.

Mutations in three loci in Dictyostelium discoideum which affect fucosylation are described. Mutations in two of these loci resulted in the simultaneous loss of two separate carbohydrate epitopes. The GA-X epitope, which was competed by L-fucose, was absent in strains carrying a modC354, modD352 or modE353 mutation. These strains exposed a new carbohydrate epitope, competed by N-acetylglucosamine, and the size of several glycoproteins was reduced. A second epitope (GA-XII) was also absent in strains carrying the modC354 or modE353 mutations, reducing the size of the glycoprotein which normally expresses it. Fucose content was reduced in the three mutants, suggesting that each mutation affected a separate step in fucosylation. The lesions did not appear to inhibit synthesis of the underlying carbohydrate, because detergent extracts of mutant vesicles were more active than normal vesicles at transferring [14C]fucose from GDP-[14C]fucose to endogenous acceptor species. The modD352 and modE353 mutant strains incorporated exogenous [3H]fucose poorly, suggesting that lesions in the modD and modE genes interfere with the biosynthesis of fucoconjugates downstream from the previously described GDP-fucose synthesis defect of the modC mutation. Intact modE353 mutant vesicles were relatively inefficient in in vitro assays, suggesting a global fucosylation defect (which is consistent with the loss of both glycoantigens, GA-X and GA-XII, in this mutant). Finally, the modC354 mutation led to delayed accumulation of slime sheath in vitro. The three genetic loci define a fucosylation pathway in D. discoideum comprising defined biochemical steps which contribute to multicellular morphogenesis in this organism.

Multiple upstream promoter elements of the gene for the pregnancy-associated tartrate-resistant acid phosphatase, uteroferrin bind human endometrial nuclear proteins.

The endometrial expression of the gene encoding porcine uteroferrin (UF), during pregnancy is presumed to be mediated by cis-regulatory regions distinct from those that confer its limited expression to other mammalian tissues and cell types. In the present study, chimeric DNA constructs of native and progressive 5' deleted promoter regions fused to the promoter chloramphenicol acetyl-transferase reporter gene were transiently transfected in the human endometrial carcinoma cell line ECC-1 to examine their ability to direct UF promoter activity. The region between -1935 and -831 bp contained negatively acting elements which drastically reduced basal promoter activity. In contrast, the region between -831 and -484 bp contributed significantly to high level basal activity. Gel retardation and footprinting assays identified factor-binding sites between -1601 and -484 bp for human endometrial nuclear proteins. One binding site corresponds to a heptamer motif (TGCTAGA) present twice within the -1601 to -831 bp region and previously shown to bind an 80 kDa porcine endometrial protein. This heptamer bound an 80 kDa nuclear protein from human ECC-1 and human Ishikawa endometrial cells and a 92 kDa protein from human placental JEG-3 cells. The other binding region within -831 to -484 bp contained GC-rich sequences, which bind human Sp1. The protected GC-rich sequence (GC-Box 1) between -768 and -749 bp also binds a 24 kDa M(r) protein. Nuclear proteins of molecular weight 40-60 kDa and distinct from Sp1, Sp2 and Sp3 bound a second GC-rich sequence (GC-Box 3) between -628 and -616 bp. These studies demonstrate that multiple elements within the UF gene promoter bind nuclear proteins which are similarly expressed in other endometrial cells and suggest that common transactivating factors may functionally mediate expression of endometrial-associated genes.

A regulatory element within the uteroferrin gene 5'-flanking region binds a pregnancy-associated uterine endometrial protein.

DNA-protein interactions within two putative regulatory regions distal from the transcription initiation site of the porcine uteroferrin (UF) gene were characterized. These regions, termed XB (-1,600 to -1,129 bp) and AB (-1,128 to -893 bp) exhibited transcriptional enhancer activities within the context of the heterologous SV40 promoter, that were specific to endometrial cells. DNase I and gel-shift assays demonstrated that both fragments contain a heptamer motif TGCTAGA that binds a nuclear protein present in crude and DEAE-fractionated nuclear extracts from porcine endometrium of pregnancy. This heptad sequence, designated as endometrial-associated sequence (EAS), is different from previously described nuclear protein-binding consensus sequences. Mutations in the heptamer motif abolished binding to the nuclear factor, as detected by gel-shift assays. The endometrial nuclear protein that interacts with the heptamer was characterized by Southwestern and UV cross-linking analysis. The protein has an approximate M(r) of 80 kD, is basic (pI 7.7-8.6) and is present in pig endometrium throughout pregnancy. The functional relevance of this DNA-binding protein in the control of UF gene transcription in the endometrium is discussed.

Non-consensus progesterone response elements mediate the progesterone-regulated endometrial expression of the uteroferrin gene.

A 2.0 kilobase-pair (kb) fragment encompassing the promoter and 5' flanking region of the uteroferrin (UF) gene was previously demonstrated to confer progesterone (P) responsiveness to chimeric UF gene promoter-reporter gene constructs when transfected in endometrial cells. In the present study, transient transfection experiments with the chloramphenicol acetyltransferase reporter gene linked to the sequentially deleted UF gene 5' flanking region and to genomic fragments within this region subcloned into the heterologous SV40 promoter were used to define the progesterone-responsive elements (PRE). The identified PREs are located distal to the promoter in the region between -1754 to -1601 bp and -893 to -678 bp of the UF gene and exhibit only limited similarities to the half-sites of the consensus palindromic PRE. The non-consensus PREs bind the progesterone receptor (PR) and independently exhibit P-dependent enhancer activities within the context of homologous and heterologous promoters in endometrial and placental cell lines. The unique features of these functional PREs suggest that formation of the P-PR complex with its cognate sequences upstream of the UF gene may be less dependent on the sequence per se but may require the binding of nuclear factors proximal to the PRE to stabilize PRE-PRE interactions.