Conserved characteristics of heterochromatin-forming DNA at the 15q11-q13 imprinting center.
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Nuclear matrix binding assays (NMBAs) define certain DNA sequences as matrix attachment regions (MARs), which often have cis-acting epigenetic regulatory functions. We used NMBAs to analyze the functionally important 15q11-q13 imprinting center (IC). We find that the IC is composed of an unusually high density of MARs, located in close proximity to the germ line elements that are proposed to direct imprint switching in this region. Moreover, we find that the organization of MARs is the same at the homologous mouse locus, despite extensive divergence of DNA sequence. MARs of this size are not usually associated with genes but rather with heterochromatin-forming areas of the genome. In contrast, the 15q11-q13 region contains multiple transcribed genes and is unusual for being subject to genomic imprinting, causing the maternal chromosome to be more transcriptionally silent, methylated, and late replicating than the paternal chromosome. We suggest that the extensive MAR sequences at the IC are organized as heterochromatin during oogenesis, an organization disrupted during spermatogenesis. Consistent with this model, multicolor fluorescence in situ hybridization to halo nuclei demonstrates a strong matrix association of the maternal IC, whereas the paternal IC is more decondensed, extending into the nuclear halo. This model also provides a mechanism for spreading of the imprinting signal, because heterochromatin at the IC on the maternal chromosome may exert a suppressive position effect in cis. We propose that the germ line elements at the 15q11-q13 IC mediate their effects through the candidate heterochromatin-forming DNA identified in this study. (+info)
The Candida albicans gene for mRNA 5-cap methyltransferase: identification of additional residues essential for catalysis.
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The 5'-cap structure of eukaryotic mRNA is methylated at the terminal guanosine by RNA (guanine-N7-)-methyltransferase (cap MTase). Saccharomyces cerevisiae ABD1 (ScABD1) and human hMet (also called CMT1) genes are responsible for this enzyme. The ABD1 homologue was cloned from the pathogenic fungus Candida albicans and named C. albicans ABD1 (CaABD1). When expressed as a fusion with glutathione S-transferase (GST), CaAbd1p displayed cap MTase activity in vitro and rescued S. cerevisiae abd1delta null mutants, indicating that CaABD1 specifies an active cap MTase. Although the human cap MTase binds to the human capping enzyme (Hce1p), which possesses both mRNA guanylyltransferase (mRNA GTase) and mRNA 5'-triphosphatase (mRNA TPase) activities, yeast two-hybrid analysis demonstrated that in yeast neither mRNA GTase nor mRNA TPase physically interacted with the Abd1 protein. Comparison of the amino acid sequences of known and putative cap MTases revealed a highly conserved amino acid sequence motif, Phe/Val-Leu-Asp/Glu-Leu/Met-Xaa-Cys-Gly-Lys-Gly-Gly-Asp-Leu-Xaa-Lys, which encompasses the sequence motif characteristic of divergent methyltransferases. Mutations in CaAbd1p of leucine at the second and the twelfth positions (so far uncharacterized) to alanine severely diminished the enzyme activity and the functionality in vivo, whereas those of leucine at the fourth, cysteine at the sixth, lysine at the eighth, and glycine at the tenth positions did not. Furthermore, valine substitution for the twelfth, but not for the second, leucine in that motif abolished the activity and functionality of CaAbd1p. Thus, it appears that leucine at the second and the twelfth positions in the motif, together with a previously identified acidic residue in the third, glycine at the sixth and glutamic acid at the eleventh positions, play important roles in the catalysis, and that side chain length is crucial for the activity at the twelfth position in the motif. (+info)
A cysteine-rich motif in poliovirus protein 2C(ATPase) is involved in RNA replication and binds zinc in vitro.
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Protein 2C(ATPase) of picornaviruses is involved in the rearrangement of host cell organelles, viral RNA replication, and encapsidation. However, the biochemical and molecular mechanisms by which 2C(ATPase) engages in these processes are not known. To characterize functional domains of 2C(ATPase), we have focused on a cysteine-rich motif near the carboxy terminus of poliovirus 2C(ATPase). This region, which is well conserved among enteroviruses and rhinoviruses displaying an amino acid arrangement resembling zinc finger motifs, was studied by genetic and biochemical analyses. A mutation that replaced the first cysteine residue of the motif with a serine was lethal. A mutant virus which lacked the second of four potential coordination sites for zinc was temperature sensitive. At the restrictive temperature, RNA replication was inhibited whereas translation and polyprotein processing, assayed in vitro and in vivo, appeared to be normal. An intragenomic second-site revertant which reinserted the missing coordination site for zinc and recovered RNA replication at the restrictive temperature was isolated. The cysteine-rich motif was sufficient to bind zinc in vitro, as assessed in the presence of 4-(2-pyridylazo)resorcinol by a colorimetric assay. Zinc binding, however, was not required for hydrolysis of ATP. 2C(ATPase) as well as its precursors 2BC and P2 were found to exist in a reduced form in poliovirus-infected cells. (+info)
Cloning of feline cDNA encoding the cytotoxic T lymphocyte-associated antigen 4 (CTLA-4).
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Cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) is a CD28 homologue which down-modulate T cell responses rather than augment them. To investigate its biological role in feline immune system, we cloned and sequenced full-length feline CTLA-4 (fCTLA-4) cDNA by RT-PCR from pokeweed mitogen stimulated peripheral blood lymphocytes. The fCTLA-4 contains an open reading frame of 669 nucleotides, coding for a polypeptide of 223 amino acids. The predicted fCTLA-4 amino acids sequence shows the homology of 86.6%, 87.0%, and 76.2% with human, bovine, and murine molecules respectively. The hexapeptide motif (MYPPPY) within the extra-cellular domain of CTLA-4 molecule, which is believed to be responsible for interaction with the B7 family members, is completely conserved in all the species. (+info)
Structure of a transcribing T7 RNA polymerase initiation complex.
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The structure of a T7 RNA polymerase (T7 RNAP) initiation complex captured transcribing a trinucleotide of RNA from a 17-base pair promoter DNA containing a 5-nucleotide single-strand template extension was determined at a resolution of 2.4 angstroms. Binding of the upstream duplex portion of the promoter occurs in the same manner as that in the open promoter complex, but the single-stranded template is repositioned to place the +4 base at the catalytic active site. Thus, synthesis of RNA in the initiation phase leads to accumulation or "scrunching" of the template in the enclosed active site pocket of T7 RNAP. Only three base pairs of heteroduplex are formed before the RNA peels off the template. (+info)
Determination of the border between the transmembrane and cytoplasmic domains of human integrin subunits.
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In this study we have determined the position of the C-terminal end of the transmembrane domains of human integrin subunits (alpha2, alpha5, beta1, beta2) in microsomal membranes using the glycosylation mapping technique. In contrast to the common view, the transmembrane helices were found to extend roughly to Phe(1129) in alpha2, to Phe(1026) in alpha5, to Ile(757) in beta1, and to His(728) in beta2. The alpha-carbon of the conserved lysine present near the C-terminal end of the transmembrane helix (Lys(1125) in alpha2, Lys(1022) in alpha5, Lys(752) in beta1, and Lys(724) in beta2) is buried in the plasma membrane, and the charged amino group most likely reaches into the polar head-group region of the lipid bilayer. A possible role for the conserved lysine in integrin function is discussed. (+info)
Activation function 2 in the human androgen receptor ligand binding domain mediates interdomain communication with the NH(2)-terminal domain.
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Activation function 2 in the ligand binding domain of nuclear receptors forms a hydrophobic cleft that binds the LXXLL motif of p160 transcriptional coactivators. Here we provide evidence that activation function 2 in the androgen receptor serves as the contact site for the androgen dependent NH(2)- and carboxyl-terminal interaction of the androgen receptor and only weakly interacts with p160 coactivators in an LXXLL-dependent manner. Mutagenesis studies indicate that it is the NH(2)-/carboxyl-terminal interaction that is required by activation function 2 to stabilize helix 12 and slow androgen dissociation critical for androgen receptor activity in vivo. The androgen receptor recruits p160 coactivators through its NH(2)-terminal and DNA binding domains in an LXXLL motif-independent manner. The results suggest a novel function for activation function 2 and a unique mechanism of nuclear receptor transactivation. (+info)
Monomeric structure of the human EphB2 sterile alpha motif domain.
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The sterile alpha motif (SAM) domain is a protein module found in many diverse signaling proteins. SAM domains in some systems have been shown to self-associate. Previous crystal structures of an EphA4-SAM domain dimer (Stapleton, D., Balan, I., Pawson, T., and Sicheri, F. (1999) Nat. Struct. Biol. 6, 44-49) and a possible EphB2-SAM oligomer (Thanos, C. D., Goodwill, K. E., and Bowie, J. U. (1999) Science 283, 833-836) both revealed large interfaces comprising an exchange of N-terminal peptide arms. Within the arm, a conserved hydrophobic residue (Tyr-8 in the EphB2-SAM structure or Phe-910 in the EphA4-SAM structure) is anchored into a hydrophobic cleft on a neighboring molecule. Here we have solved a new crystal form of the human EphB2-SAM domain that has the same overall SAM domain fold yet has no substantial intermolecular contacts. In the new structure, the N-terminal peptide arm of the EphB2-SAM domain protrudes out from the core of the molecule, leaving both the arm (including Tyr-8) and the hydrophobic cleft solvent-exposed. To verify that Tyr-8 is solvent-exposed in solution, we made a Tyr-8 to Ala-8 mutation and found that the EphB2-SAM domain structure and stability were only slightly altered. These results suggest that Tyr-8 is not part of the hydrophobic core of the EphB2-SAM domain and is conserved for functional reasons. Cystallographic evidence suggests a possible role for the N-terminal arm in oligomerization. In the absence of a direct demonstration of biological relevance, however, the functional role of the N-terminal arm remains an open question. (+info)