Cloning and characterization of the 5' end (exon 1) of the gene encoding human factor X

Human blood-coagulation factor X (hBCFX) is a serine protease zymogen which participates in the middle phase of blood coagulation. Recently, we and others have reported the cDNA sequences. At present, partial hBCFX gene structure is available. In this paper, we report the isolation of two genomic clones, the X-emb lambda phage clone encoding exons 1 and 2 of the hBCFX gene, and the X-cos cosmid clone encoding exons 2-8. The restriction map of the hBCFX region spans 55 kb. The gene itself was found to be 27 kb long. The sequence of the 5' region of the hBCFX gene has been determined and reveals an ATTTG pentanucleotide, which also occurs in a similar location in the genes encoding factors VII, IX, protein CC and C prothrombin, suggesting that this motif might be of importance in the regulation of these genes.     

TGGCA protein is present in erythroid nuclei and binds within the nuclease-hypersensitive sites 5' of the chicken beta H- and beta A-globin genes

The developmentally regulated 5'-flanking DNase-I-hypersensitive site of the chicken beta H-globin gene in nuclei contains a subregion which is resistant to DNase I and which disappears when nuclei are extracted with 0.3 M NaCl, suggesting that there are salt-extractable proteins bound to sequences within this region. The 0.3 M NaCl extract contains two proteins which bind in vitro to these sequences. One of the binding sequences has an inverted repeat very similar to that bound by TGGCA protein. Partially purified TGGCA protein from chicken liver binds to this sequence in vitro giving exactly the same footprint as that obtained with erythroid nuclear proteins. Similarly TGGCA protein binds to an inverted repeat with the beta A-globin 5'-hypersensitive site giving a footprint identical to that obtained with erythroid nuclear protein extracts. From competition footprinting experiments and the electrophoretic mobility of the protein-DNA complex, it is concluded that the erythroid proteins previously described as binding to the beta H- and beta A-globin inverted repeats within the 5'-flanking hypersensitive sites both belong to the TGGCA protein family.     

The bovine leukemia virus encapsidation signal is discontinuous and extends into the 5' end of the gag gene.

In order to define bovine leukemia virus (BLV) sequences required for efficient vector replication, a series of mutations were made in a BLV vector. Testing the replication efficiency of the vectors with a helper virus and helper plasmids allowed for separation of the mutant vectors into three groups. The replication efficiency of the first group was reduced by a factor of 7; these mutants contained deletions in the 5' end of the gag gene. The second group of mutants had replication reduced by a factor of 50 and had deletions including the 5' untranslated leader region. The third group of mutants replicated at levels comparable to those of the parental vector and contained deletions of the 3' end of the gag gene, the pol gene, and the env gene. Analysis of cytoplasmic and virion RNA levels indicated that vector RNA expression was not affected but that the vector RNA encapsidation was less efficient for group 1 and group 2 mutants. Additional mutations revealed two regions important for RNA encapsidation. The first region is a 132-nucleotide-base sequence within the gag gene (nucleotides 1015 to 1147 of the proviral DNA) and facilitates efficient RNA encapsidation in the presence of the second region. The second region includes a 147-nucleotide-base sequence downstream of the primer binding site (nucleotide 551) and near the gag gene start codon (nucleotide 698; gag begins at nucleotide 628) and is essential for RNA encapsidation. We conclude that the encapsidation signal is discontinuous; a primary signal, essential for RNA encapsidation, is largely in the untranslated leader region between the primer binding site and near the gag start codon. A secondary signal, which facilitates efficient RNA encapsidation, is in a 132-nucleotide-base region within the 5' end of the gag gene.     

An RNA pseudoknot is an essential structural element of the internal ribosome entry site located within the hepatitis C virus 5' noncoding region.

Translation of the human hepatitis C virus (HCV) RNA genome occurs by a mechanism known as "internal ribosome entry." This unusual strategy of translation is employed by naturally uncapped picornaviral genomic RNAs and several cellular mRNAs. A common feature of these RNAs is a relatively long 5' noncoding region (NCR) that folds into a complex secondary structure harboring an internal ribosome entry site (IRES). Evidence derived from the use of dicistronic expression systems, combined with an extensive mutational analysis, demonstrated the presence of an IRES within the HCV 5'NCR. The results of our continued mutational analysis to map the critical structural elements of the HCV IRES has led to the identification of a pseudoknot structure upstream of the initiator AUG. The evidence presented in this study is based upon the mutational analysis of the putative pseudoknot structure. This is further substantiated by biochemical and enzymatic probing of the wild-type and mutant 5'NCR. Further, the thermodynamic calculations, based upon a modified RNAKNOT program, are consistent with the presence of a pseudoknot structure located upstream of the initiator AUG. Maintenance of this structural element is critical for internal initiation of translation. The pseudoknot structure in the 5'NCR represents a highly conserved feature of all HCV subtypes and members of the pestivirus family, including hog cholera virus and bovine viral diarrhea virus.     

Reevaluation of the conclusion that IRES-activity reported within the 5' leader of the TIF4631 gene is due to promoter activity

We previously reported that the 5' leader of the mRNA-encoding initiation factor eIF4G in Saccharomyces cerevisiae can function as a translational enhancer and as an internal ribosome entry site (IRES) when tested in cells. However, Verge and colleagues recently suggested that this sequence does not facilitate translation initiation, but inhibits translation in vitro and has promoter activity when tested in cells. We disagree with these conclusions and respond by showing that the data are most consistent with an internal initiation mechanism.     

Differences in changes of the alpha1-beta2 subunit contacts between ligand binding to the alpha and beta subunits of hemoglobin A: UV resonance raman analysis using Ni-Fe hybrid hemoglobin

The alpha1-beta2 subunit contacts in the half-ligated hemoglobin A (Hb A) have been explored with ultraviolet resonance Raman (UVRR) spectroscopy using the Ni-Fe hybrid Hb under various solution conditions. Our previous studies demonstrated that Trpbeta37, Tyralpha42, and Tyralpha140 are mainly responsible for UVRR spectral differences between the complete T (deoxyHb A) and R (COHb A) structures [Nagai, M., Wajcman, H., Lahary, A., Nakatsukasa, T., Nagatomo, S., and Kitagawa, T. (1999) Biochemistry, 38, 1243-1251]. On the basis of it, the UVRR spectra observed for the half-ligated alpha(Ni)beta(CO) and alpha(CO)beta(Ni) at pH 6.7 in the presence of IHP indicated the adoption of the complete T structure similar to alpha(Ni)beta(deoxy) and alpha(deoxy)beta(Ni). The extent of the quaternary structural changes upon ligand binding depends on pH and IHP, but their characters are qualitatively the same. For alpha(Ni)beta(Fe), it is not until pH 8.7 in the absence of IHP that the Tyr bands are changed by ligand binding. The change of Tyr residues is induced by binding of CO, but not of NO, to the alpha heme, while it was similarly induced by binding of CO and NO to the beta heme. The Trp bands are changed toward R-like similarly for alpha(Ni)beta(CO) and alpha(CO)beta(Ni), indicating that the structural changes of Trp residues are scarcely different between CO binding to either the alpha or beta heme. The ligand induced quaternary structural changes of Tyr and Trp residues did not take place in a concerted way and were different between alpha(Ni)beta(CO) and alpha(CO)beta(Ni). These observations directly indicate that the phenomenon occurring at the alpha1-beta2 interface is different between the ligand binding to the alpha and beta hemes and is greatly influenced by IHP. A plausible mechanism of the intersubunit communication upon binding of a ligand to the alpha or beta subunit to the other subunit and its difference between NO and CO as a ligand are discussed.     

Regional methylation of the 5' end CpG island of BRCA1 is associated with reduced gene expression in human somatic cells.

In mammalians, demethylation of specific promoter regions often correlates with gene activation; inversely, dense methylation of CpG islands leads to gene silencing, probably mediated by methyl-CpG binding proteins. In cell lines and cancers, inhibition of tissue-specific genes and tumor suppressor genes expression seems to be related to such hypermethylation. The 5' end of the breast cancer predisposition gene BRCA1 is embedded in a large CpG island of approximately 2.7 kb in length. In human sporadic breast cancers, the down-regulation of BRCA1 does not seem to be related to BRCA1 gene alterations. Southern blot analysis and the bisulfite sequencing method indicate that the BRCA1 CpG island is regionally methylated in all human tissues analyzed and unmethylated in the gametes, suggesting a role for DNA methylation in the control of gene expression. We have therefore investigated the potential role of methyl-CpG binding proteins in the regulation of BRCA1 gene expression. In vitro, partial methylation of constructs containing this region strongly inhibits gene expression in the presence of MeCP2 protein. Moreover, in the five human cell lines analyzed, chemically induced hypomethylation is associated with BRCA1 gene activation. These data suggest that methyl-CpG binding proteins might be associated with the control of BRCA1 gene expression and that methyl-DNA binding proteins may participate in the regulation of gene expression in mammalian cells.     

Cryptic splice site usage in exon 7 of the human fibrinogen Bbeta-chain gene is regulated by a naturally silent SF2/ASF binding site within this exon

In this work we report the identification of a strong SF2/ASF binding site within exon 7 of the human fibrinogen Bbeta-chain gene (FGB). Its disruption in the wild-type context has no effect on exon recognition. However, when the mutation IVS7 + 1G>T--initially described in a patient suffering from congenital afibrinogenemia--is present, this SF2/ASF binding site is critical for cryptic 5'ss (splice site) definition. These findings, besides confirming and extending previous results regarding the effect of SF2/ASF on cryptic splice site activation, identify for the first time an enhancer sequence in the FGB gene specific for cryptic splice site usage. Taken together, they suggest the existence of a splicing-regulatory network that is normally silent in the FGB natural splicing environment but which can nonetheless influence splicing decisions when local contexts allow. On a more general note, our conclusions have implications for the evolution of alternative splicing processes and for the development of methods to control aberrant splicing in the context of disease-causing mutations.     

The 5' end of U2 snRNA is in close proximity to U1 and functional sites of the pre-mRNA in early spliceosomal complexes

Recognition and pairing of the correct 5' and 3' splice sites (ss) of a pre-mRNA are critical events that occur early during spliceosome assembly. Little is known about the spatial organization in early spliceosomal complexes of the U1 and U2 snRNPs, which together with several non-snRNP proteins, are involved in juxtapositioning the functional sites of the pre-mRNA. To better understand the molecular mechanisms of splice-site recognition/pairing, we have examined the organization of U2 relative to U1 and pre-mRNA in spliceosomal complexes via hydroxyl-radical probing with Fe-BABE-tethered U2 snRNA. These studies reveal that functional sites of the pre-mRNA are located close to the 5' end of U2 both in E and A complexes. U2 is also positioned close to U1 in a defined orientation already in the E complex, and their relative spatial organization remains largely unchanged during the E to A transition.     

Tissue restricted and stage specific transcription is maintained within 411 nucleotides flanking the 5'' end of the chicken alpha-skeletal actin gene.

alpha-skeletal actin message levels have been shown to be tightly regulated in chicken primary myoblast cultures. To test for gene elements required for muscle cell specific expression, DNA sequences containing the 5'-flanking regions of the chicken alpha-skeletal actin, beta-cytoplasmic actin, and the histone H2b genes were linked to the coding sequences of the chloramphenicol acetyltransferase gene and transfected into myogenic and non-myogenic cells. In contrast to beta-actin CAT hybrids, the alpha-skeletal actin CAT constructions displayed restricted CAT expression in transfected non-myogenic cells. We showed that a 411 nucleotide fragment flanking the 5' end of of the alpha-skeletal actin gene was responsible for a 9-15 fold increase in CAT enzymatic activity during myoblast fusion, versus only a transient 2 fold rise for the beta-actin and histone flanking sequences. These results indicate that DNA sequences within 411 bp of the 5' terminus of the alpha-skeletal actin gene influenced its cell type and stage specific expression.