Interaction of cis-regulatory element in the 5' flanking sequence of human beta-globin gene with trans-acting factors at different developmental stages

Human beta-globin gene mainly expressed in the adult bone marrow, while not expressed in fetal liver, adult liver and K562 cells. Using gel mobility shift assay, different protein factors binding to the regulatory elements (from -372 to -194 bp) in the 5' flanking sequence of human beta-globin gene were detected in the nuclear extracts from human fetal liver, adult liver and K562 cells respectively. Competitive experiments showed that both the protein factors from adult and K562 cells not only bind to negative control region 2 (NCR 2, from -372 to -224 bp), but also bind to positive control region (PCR, from -223 to -194 bp), suggesting that there may be similar mechanism to silence the expression of human beta-globin gene in these two kind of cells. The protein factor in human fetal liver only binds to NCR 2, indicating that there is a particular silencing mechanism for beta-globin gene expression in human fetal stage.     

Interactions of rat repetitive sequence MspI8 with nuclear matrix proteins during spermatogenesis

Using the Southwestern blot analysis we have studied the interactions between rat repetitive sequence MspI8 and the nuclear matrix proteins of rat testis cells. Starting from 2 weeks the young to adult animals showed differences in type of testis nuclear matrix proteins recognizing the MspI8 sequence. The same sets of nuclear matrix proteins were detected in some fractions enriched in spermatocytes and spermatides and obtained after fractionation of testis cells of adult animals by the velocity sedimentation technique.     

Nuclear proteins from liver and kidney bind a 37 bp sequence in the 5' upstream region of the mGAT1 gene

The mouse GABA transporter (mGAT1) gene has been shown to be exclusively expressed in brain by Northern and Western blot analyses. The interactions between the 5' flanking region of the mGAT1 gene and nuclear proteins from different mouse tissues were studied by means of gel-shift assay. Our results show that nuclear protein factors from non-nervous tissues can specifically recognize a 37 bp sequence that is conserved in the 5' flanking region between the human and mouse GAT1 genes. Similar nuclear protein factors were also found to exist in rat, rabbit and pig.     

A mutation in the flanking 5'-TA-3' dinucleotide prevents excision of an internal eliminated sequence from the Paramecium tetraurelia genome

The germline chromosomes in Paramecium and other ciliated protozoa contain regions of DNA that are excised and eliminated during the development of a new macronuclear genome. Paramecium tetraurelia internal eliminated sequences (IESs) are invariably flanked by a 5'-TA-3' dinucleotide sequence that is part of a larger 8-bp terminal inverted-repeat consensus sequence. Both features, the absolutely conserved 5'-TA-3' and the remaining 6-bp terminal inverted repeat, are shared with the mariner/Tc1 class of transposons. In this article we describe a mutant cell line (AIM-2) defective in excision of a single IES from the coding region of the A51 surface antigen gene. Excision of the 370-bp IES6649 is prevented by a single A to G transition in the invariably conserved 5'-TA-3' dinucleotide. Failure to excise IES6649 also revealed a 29-bp IES located inside IES6649. Additional experiments with the previously isolated AIM-1 mutant, which also contains an internal IES, shows that alternate excision using the wild-type end of IES2591 with an end from the internal IES is extremely rare or nonexistent. These results indicate that IESs are discrete elements whose excision depends upon nucleotides located within the consensus sequence, but also suggest that additional information is required to match one end of an IES with its excision partner.     

The U1 small nuclear ribonucleoprotein/5' splice site interaction affects U2AF65 binding to the downstream 3' splice site

In the gene of the neural cell adhesion molecule, the 5' splice site of the alternate exon 18 plays an important role in establishing regulated splicing profiles. To understand how the 5' splice site of exon 18 contributes to splicing regulation, we have investigated the interaction of the U2AF65 splicing factor to pre-mRNAs that contained portions of the constitutive exon 17 or the alternate exon 18 fused to exon 19 and separated by a shortened intron. Despite sharing an identical 3' splice site, only the pre-mRNA that contained a portion of exon 17 and its associated 5' splice site displayed efficient U2AF65 cross-linking. Strikingly, a G-->U mutation at position +6 of the intron, converting the 5' splice site of exon 18 into that of exon 17, stimulated U2AF65 crosslinking. The improved cross-linking efficiency of U2AF65 to a pre-mRNA carrying the 5' splice site of exon 17 required the integrity of the 5' end of U1 but not of U2 small nuclear RNA. Our results indicate that neural cell adhesion molecule 5' splice site sequences influence U2AF65 binding through a U1 small nuclear ribonucleoprotein/U2AF interaction that occurs at the commitment stage of spliceosome assembly, before stable binding of the U2 small nuclear ribonucleoprotein. Thus, the 5' splice sites of exons 17 and 18 differentially affect U2AF65 binding to the 3' splice site of exon 19. Factors that modulate U1 small nuclear ribonucleoprotein binding to these 5' splice sites may play a critical role in regulating exon 18 skipping.     

Studies on the binding of adenylyl-3', 5'-cytidine to ribonuclease

The interaction of adenylyl-3',5'-cytidine (ApC) with ribonuclease-A (RNAase-A) was studied by steady-state kinetics and ultraviolet difference spectroscopy. X-ray difference Fourier synthesis at 4 A resolution was also used to study the binding of ApC to RNAase-S. Unlike well-studied compounds like uridylyl-3',5'-adenosine, ApC binds in an unique way: (1) the cytidine moiety is bound to the B1 and R1 sites, (2) the adenosine moiety protrudes to the solution and is not fixed spatially and (3) the phosphate group is bound to the non-specific site (the "Po site") previously postulated (Sawada, F. and Irie, M. (1969) J. Biochem. (Tokyo) 66, 415--418) as the binding site for the 5'-phosphate of uridine 2',5'-diphosphate or uridine 3',5'-diphosphate. This conclusion is consistent with that derived for adenylyl-3',5' -4-thiouridine based on CD difference spectroscopy (White, M.D., Keren-Zur, M. and Lapidot, Y. (1977) Nucleic Acid Res. 4, 843--851). The "Po site" is most likely the epsilon-amino group of Lys 66.     

Localization of human cytomegalovirus structural proteins to the nuclear matrix of infected human fibroblasts

The intranuclear assembly of herpesvirus subviral particles remains an incompletely understood process. Previous studies have described the nuclear localization of capsid and tegument proteins as well as intranuclear tegumentation of capsid-like particles. The temporally and spatially regulated replication of viral DNA suggests that assembly may also be regulated by compartmentalization of structural proteins. We have investigated the intranuclear location of several structural and nonstructural proteins of human cytomegalovirus (HCMV). Tegument components including pp65 (ppUL83) and ppUL69 and capsid components including the major capsid protein (pUL86) and the small capsid protein (pUL48/49) were retained within the nuclear matrix (NM), whereas the immediate-early regulatory proteins IE-1 and IE-2 were present in the soluble nuclear fraction. The association of pp65 with the NM resisted washes with 1 M guanidine hydrochloride, and direct binding to the NM could be demonstrated by far-Western blotting. Furthermore, pp65 exhibited accumulation along the nuclear periphery and in far-Western analysis bound to proteins which comigrated with proteins of the size of nuclear lamins. A direct interaction between pp65 and lamins was demonstrated by coprecipitation of lamins in immune complexes containing pp65. Together, our findings provide evidence that major virion structural proteins localized to a nuclear compartment, the NM, during permissive infection of human fibroblasts.     

Evidence for the involvement of arginine 462 and the flanking sequence of human C4 beta-chain in mediating C5 binding to the C4b subcomponent of the classical complement pathway C5 convertase

Replacement of human C4 beta-chain residue arginine 458 by tryptophan, a substitution that occurs naturally in the hemolytically inactive A6 allotype of C4, totally abrogates the molecule's ability to act as a C5 binding subunit of the classical pathway C5 convertase. Hydropathy plots predict R458 to be within a hydrophilic segment extending from residue 455 to 469 and having the sequence SIERPDSRPPRVGDT. To further assess the potential involvement of this segment in the C5 binding function of C4, we have engineered "ala-scan" mutants through this segment, concentrating predominantly on charged residues, and analyzed their functional profiles. C4B isotype mutant proteins S455A (0.7), E457A (1.1), R458A (0.3), D460A (0.2), R462A (0.0), R465A (0.6), and D468A (0.3) displayed the relative to wild-type hemolytic activities indicated in the parentheses. In all cases, the hemolytic defect was accounted for solely at the C5 convertase stage. The total absence of C5 binding activity in the R462A mutant suggests a requirement for the guanidinium group per se, because mutants with a charge-conservative lysine or a relatively isosteric methionine at this position were also completely inactive. In contrast, the inactivity of the C4A6-like R458W mutant is probably caused by the intolerance of tryptophan in a hydrophilic segment, as substitution of R458 by alanine or methionine yielded recombinant molecules that retained 30% and 60% of wild-type hemolytic activity, respectively. Taken together, the mutagenesis results strongly imply that residues in the 455-469 segment contribute to the C5 binding site in C4; however, the conformational context of the segment appears to be crucial, as a synthetic peptide corresponding to this segment displayed no ability to inhibit C5 binding to surface-bound C4b.     

Species-specific binding of sperm proteins to the extracellular matrix (zona pellucida) of the egg

The zona pellucida is an extracellular matrix surrounding the mammalian egg where species-specific gamete recognition and signaling occur. Pig zona pellucida were isolated in large amounts and used as an affinity matrix for detergent-solubilized, biotinylated membrane proteins of pig spermatozoa. On non-reducing SDS-polyacrylamide gel electrophoresis, specifically bound sperm proteins migrated with M(r) 170,000, 150,000, 130,000, 56,000, and 50,000 (p50). Disulfide bond reduction separated each of the M(r) 130,000-170,000 proteins into M(r) 105,000 (p105) and M(r) 45,000 (p45) subunits, indicating that these high M(r) proteins are related. Based on two-dimensional electrophoresis, the M(r) 56,000 band was composed of three to four proteins that migrated with M(r) 56,000-62,000 (p56-62) in the second (reducing) dimension. p50 bound to heterologous zona pellucida (murine, bovine) and to Xenopus laevis oocyte envelopes, demonstrating a lack of species specificity to its binding and was identified as proacrosin/acrosin based on amino acid sequences of two tryptic peptides and its interaction with monospecific antibodies to proacrosin. In contrast, p105/p45 and one or more of the p56-62 proteins bound to pig zona pellucida but not to the egg extracellular matrices of the other species; these proteins therefore exhibited the species-specific binding to the zona pellucida expected for molecules involved in specific gamete adhesion. Amino acid sequences of nine tryptic peptides derived from p105/p45 did not match peptide sequences in existing databases, establishing it as a unique protein. These (p105/p45 and at least one p56-62 protein) are the first sperm membrane proteins to be identified that bind in a species-specific manner to the egg extracellular matrix.     

Structural basis for binding of the plasmid ColIb-P9 antisense Inc RNA to its target RNA with the 5'-rUUGGCG-3' motif in the loop sequence

The sequence 5'-rUUGGCG-3' is conserved within the loop regions of antisense RNAs or their targets involved in replication of various prokaryotic plasmids. In IncIalpha plasmid ColIb-P9, the partially base paired 21-nucleotide loop of a stem-loop called structure I within RepZ mRNA contains this hexanucleotide sequence, and comprises the target site for the antisense Inc RNA. In this report, we find that the base pairing interaction at the 5'-rGGC-3' sequence in the hexanucleotide motif is important for interaction between Inc RNA and structure I. In addition, the 21-base loop domain of structure I is folded tighter than predicted, with the hexanucleotide sequence at the top. The second U residue in the sequence is favored for Inc RNA binding in a base-specific manner. On the other hand, the upper domain of the Inc RNA stem-loop is loosely structured, and maintaining the loop sequence single-stranded is important for the intermolecular interaction. Based on these results, we propose that a structural feature in the loop I domain, conferred probably by the conserved 5'-rUUGGCG-3' sequence, favors binding to a complementary, single-stranded RNA. This model also explains how the RepZ mRNA pseudoknot, described in the accompanying paper (Asano, K., and Mizobuchi, K. (1998) J. Biol. Chem. 273, 11815-11825) is formed specifically with structure I. A possible conformation adopted by the 5'-rUUGGCG-3' loop sequence is discussed.     

Effect of protein aggregation in the aqueous phase on the binding of membrane proteins to membranes

Analysis of the binding of hydrophobic peptides or proteins to membranes generally assumes that the solute is monomeric in both the aqueous phase and the membrane. Simulations were performed to examine the effect of solute self-association in the aqueous phase on the binding of monomeric solute to lipid vesicles. Aggregation lowered the initial concentration of monomeric solute, which was then maintained at a relatively constant value at the expense of the aggregated solute, as the lipid concentration was increased. The resultant binding isotherm has a more linear initial portion rather than the classic hyperbolic shape. Although this shape is diagnostic of solute self-association in the aqueous phase, various combinations of values for the membrane partition coefficient and the solute self-association constant will generate similar isotherms. Data for cytochrome b5 were analyzed and, when the self-association constant was estimated by gel filtration, a unique value for the membrane partition coefficient was obtained. Thus, to obtain a true partition coefficient the state of the solute in the aqueous phase must be known. If the concentration of the monomeric solute species in the aqueous phase can be independently determined, then, even with heterogeneous aggregates, the true partition coefficient can be obtained.