Orc mutants arrest in metaphase with abnormally condensed chromosomes.

The origin recognition complex (ORC) is a six subunit complex required for eukaryotic DNA replication initiation and for silencing of the heterochromatic mating type loci in Saccharomyces cerevisiae. Our discovery of the Drosophila ORC complex concentrated in the centric heterochromatin of mitotic cells in the early embryo and its interactions with heterochromatin protein 1 (HP-1) lead us to speculate that ORC may play some general role in chromosomal folding. To explore the role of ORC in chromosomal condensation, we have identified a mutant of subunit 5 of the Drosophila melanogaster origin recognition complex (Orc5) and have characterized the phenotypes of both the Orc5 and the previously identified Orc2 mutant, k43. Both Orc mutants died at late larval stages and surprisingly, despite a reduced number of S-phase cells, an increased fraction of cells were also detected in mitosis. For this latter population of cells, Orc mutants arrest in a defective metaphase with shorter and thicker chromosomes that fail to align at the metaphase plate within a poorly assembled mitotic spindle. In addition, sister chromatid cohesion was frequently lost. PCNA and MCM4 mutants had similar phenotypes to Orc mutants. We propose that DNA replication defects trigger the mitotic arrest, due to the fact that frequent fragmentation was observed. Thus, cells have a mitotic checkpoint that senses chromosome integrity. These studies also suggest that the density of functional replication origins and completion of S phase are requirements for proper chromosomal condensation.

Association of the origin recognition complex with heterochromatin and HP1 in higher eukaryotes.

The origin recognition complex (ORC) is required to initiate eukaryotic DNA replication and also engages in transcriptional silencing in S. cerevisiae. We observed a striking preferential but not exclusive association of Drosophila ORC2 with heterochromatin on interphase and mitotic chromosomes. HP1, a heterochromatin-localized protein required for position effect variegation (PEV), colocalized with DmORC2 at these sites. Consistent with this localization, intact DmORC and HP1 were found in physical complex. The association was shown biochemically to require the chromodomain and shadow domains of HP1. The amino terminus of DmORC1 contained a strong HP1-binding site, mirroring an interaction found independently in Xenopus by a yeast two-hybrid screen. Finally, heterozygous DmORC2 recessive lethal mutations resulted in a suppression of PEV. These results indicate that ORC may play a widespread role in packaging chromosomal domains through interactions with heterochromatin-organizing factors.

The DNA-binding domain of the hexameric arginine repressor.

The arginine repressor of Escherichia coli is a classical feedback regulator, signalling the availability of L-arginine inside the cell. It differs from most other bacterial repressors in functioning as a hexamer, but structural details have been lacking and its shares no clear sequence homologies with other transcriptional regulators. Analysis of the amino acid residue sequence and proteolytic cleavage pattern of the repressor was used to identify a region predicted to house the DNA-binding function. When this protein fragment is overexpressed from a clone of the corresponding gene fragment, it represses ornithine transcarbamylase levels in vivo, and binds to the operator DNA in vitro, both in an arginine-independent manner. Sedimentation equilibrium and gel filtration indicate that the purified protein fragment is a monomer in solution. The results thus define the domain organization of the repressor at low resolution, suggesting that the N and C-terminal portions of the polypeptide chain are separated by a structural and functional border that decouples hexamerization and arginine binding from DNA binding.

Interaction between the two conserved single-stranded regions at the decoding site of small subunit ribosomal RNA is essential for ribosome function.

Formation of the tertiary base pair G1401:C1501, which brings together two universally present and highly sequence-conserved single-stranded segments of small subunit ribosomal RNA, is essential for ribosome function. It was previously reported that mutation of G1401 inactivated all in vitro functions of the ribosome [Cunningham et al. (1992) Biochemistry 31, 7629-7637]. Here we show that mutation of C1501 to G was equally inactivating but that the double mutant C1401:G1501 with the base pair reversed had virtually full activity for tRNA binding to the P, A, and I sites and for peptide bond formation. Initiation-dependent formation of the first peptide bond remained 70-85% inhibited, despite full 70S initiation complex formation ability as evidenced by the ability to form fMET-puromycin. These results suggest that the defect in formation of the first peptide bond lies in filling the initial A site, Ai, rather than the subsequent elongation A sites, Ae. An increased mobility around the anticodon was detected by UV cross-linking of the anticodon of P-site-bound tRNA to C1399 as well as to the expected C1400. These findings provide the first experimental evidence for the existence of the G1401:C1501 base pair and show that this base pair, located at the decoding site, is essential for function. The structural implications of tertiary base pair formation are discussed.

A heme binding site on myelin basic protein: characterization, location, and significance.

Myelin basic protein (MBP), an extrinsic membrane protein from the myelin sheath, binds dicyanohemin. The binding generates absorption bands in the Soret region and quenches the fluorescence emitted by the sole tryptophan residue. The absorption titration curves in the Soret demonstrate that the binding is stoichiometric, one heme per protein, with a large value of the extinction coefficient (8 X 10(4) M-1 cm-1 at 420 nm). Fluorescence quenching titration curves indicate an identical stoichiometry and a low quenching efficiency of 20%. From the heme titration curve the association constant between dicyanohemin and MBP is estimated to be greater than or equal to 10 nM-1 in 50 mM 4-morpholinepropanesulfonic acid buffer, pH 7.0, at 20 degrees C. Digestion of MBP by Staphylococcus aureus V8 protease yields a peptide (38-118) whose heme binding properties are identical to those of MBP. In contrast, peptides obtained by digestion of MBP with cathepsin D do not exhibit any specific binding of dicyanohemin. The cleavage of the Phe-Phe (42-43) bond appears to be critical in this respect. A comparison of the sequence immediately preceding, including these residues with a probable heme binding site of a mitochondrial cytochrome b, reveals a high degree of homology. The possible significance of heme binding is discussed.

Subgroups of amino acid sequences in the variable regions of immunoglobulin heavy chains.

The amino acid sequence of the first 133 residues of the heavy (gamma) chain from a human gammaG immunoglobulin (He) has been determined. This gamma-chain is identical in Gm type to that of protein Eu, the complete sequence of which has been reported. Comparison of the two sequences substantiates the previous suggestion that there are subgroups of variable regions of heavy chains. The variable region of Eu has been assigned to subgroup I and that of He to subgroup II; on the other hand, the constant regions of the two proteins appear to be identical. Comparison of the sequence of the heavy chain of He with the heavy chain sequences determined in other laboratories suggests that the variable region of subgroup II is at least 118 residues long. The nature and distribution of amino acid variations in this heavy chain subgroup resemble those observed in light chain subgroups. These studies provide evidence that the translocation hypothesis applies to heavy as well as to light chains, viz., genes for variable regions (V) are somatically translocated to genes for constant regions (C) to form complete VC structural genes.