Chromosomal localization of several families of repetitive sequences by in situ hybridization.

Four recombinant DNA clones (H1, H7, H12, and H15) carrying low-repetitive human DNA were previously isolated from a human genomic library based on their specificity for chromosome 21 and were studied for their distribution as determined by in situ hybridization. Clone H7 hybridized to the satellite regions of chromosomes 13, 14, 15, 21, and 22 as well as to the centromere region of chromosome 1. Clone H12 hybridized strongly to chromosomes 11 and 17 and the centromere of the X. Clones H1 and H15 had a very widespread distribution throughout the genome. Clone H15 hybridized significantly more to the short arm of chromosome 18 than to any other chromosomal segment. Clone H1 hybridized strongly to the centromere of chromosome 19 and also showed random distribution on all the other human chromosomes. We conclude that these probes appear to represent four repetitive families that demonstrate in situ hybridization patterns that do not correspond with those of any other repetitive family. Further, the in situ hybridization patterns do not show the strong chromosome 21 specificity originally defined by Southern blot analysis. The nature and chromosomal localization of these repetitive families should be useful in regional mapping and evolutionary studies and give additional insight into chromosomal organization.     

A major difference between the divergence patterns within the lines-1 families in mice and voles

L1 retroposons are represented in mice by subfamilies of interspersed sequences of varied abundance. Previous analyses have indicated that subfamilies are generated by duplicative transposition of a small number of members of the L1 family, the progeny of which then become a major component of the murine L1 population, and are not due to any active processes generating homology within preexisting groups of elements in a particular species. In mice, more than a third of the L1 elements belong to a clade that became active approximately 5 Mya and whose elements are > or = 95% identical. We have collected sequence information from 13 L1 elements isolated from two species of voles (Rodentia: Microtinae: Microtus and Arvicola) and have found that divergence within the vole L1 population is quite different from that in mice, in that there is no abundant subfamily of homologous elements. Individual L1 elements from voles are very divergent from one another and belong to a clade that began a period of elevated duplicative transposition approximately 13 Mya. Sequence analyses of portions of these divergent L1 elements (approximately 250 bp each) gave no evidence for concerted evolution having acted on the vole L1 elements since the split of the two vole lineages approximately 3.5 Mya; that is, the observed interspecific divergence (6.7%-24.7%) is not larger than the intraspecific divergence (7.9%-27.2%), and phylogenetic analyses showed no clustering into Arvicola and Microtus clades.      

A data management system for structural genomics

Background  

Structural genomics (SG) projects aim to determine thousands of protein structures by the development of high-throughput techniques for all steps of the experimental structure determination pipeline. Crucial to the success of such endeavours is the careful tracking and archiving of experimental and external data on protein targets.     

心脏特异表达LMNA^E82K转基因小鼠的建立

目的建立心脏特异表达LMNAE82K转基因小鼠,为研究LMNAE82K与心肌病发病机制的关系提供工具动物。方法把LMNAE82K基因插入α-MHC启动子下游,构建转基因表达载体,显微注射法建立C57BL/6JLMNAE82K转基因小鼠,PCR鉴定转基因小鼠的基因型,采用Western Blot鉴定LMNAE82K在心脏组织中的表达,H＆E染色和超声检测转基因小鼠心脏的病理改变。结果建立了2个心脏组织特异表达LMNAE82K的转基因小鼠品系。超声检查显示转基因小鼠心室壁变薄,收缩期容积和舒张期容积增加,射血分数及短轴缩短率降低。结论LMNAE82K转基因小鼠具有LMNAE82K引起的家族性扩心病有类似的病理变化,为研究LMNAE82K与心肌病发病机制的关系的研究提供了有价值的疾病动物模型。     

Molecular Mechanisms of Bitopic Ligand Engagement with the M1 Muscarinic Acetylcholine Receptor

TBPB and 77-LH-28-1 are selective agonists of the M₁ muscarinic acetylcholine receptor (mAChR) that may gain their selectivity through a bitopic mechanism, interacting concomitantly with the orthosteric site and part of an allosteric site. The current study combined site-directed mutagenesis, analytical pharmacology,and molecular modeling to gain further insights into the structural basis underlying binding and signaling by these agonists. Mutations within the orthosteric binding site caused similar reductions in affinity and signaling efficacy for both selective and prototypical orthosteric ligands. In contrast, the mutation of residues within transmembrane helix (TM) 2 and the second extracellular loop (ECL2) discriminated between the different classes of ligand. In particular, ECL2 appears to be involved in the selective binding of bitopic ligands and in coordinating biased agonism between intracellular calcium mobilization and ERK1/2 phosphorylation. Molecular modeling of the interaction between TBPB and the M₁ mAChR revealed a binding pose predicted to extend from the orthosteric site up toward a putative allosteric site bordered by TM2, TM3, and TM7, thus consistent with a bitopic mode of binding. Overall, these findings provide valuable structural and mechanistic insights into bitopic ligand actions and receptor activation and support a role for ECL2 in dictating the active states that can be adopted by a G protein-coupled receptor. This may enable greater selective ligand design and development for mAChRs and facilitate improved identification of bitopic ligands.