In addition to sustaining an exponentially increasing rate of gene finding (Collins 1995), yeast artificial chromosome/sequence-tagged site (STS/YAC)-based maps (Burke et al. 1987;Olson et al. 1989) have begun to reveal additional features of chromosome structure and dynamics. For example, during the development of maps for subportions of the X chromosome, the existence of a second "pseudoautosomal" region at the Xq terminus of the chromosome was demonstrated (Freije and Schlessinger 1992;Li and Hamer 1995), followed by the discovery that the region shows a unique phenomenon of gene inactivation on both the X and Y homologs (D'Esposito et al. 1996). In another instance, it was shown that a cluster of genes in a delimited segment of XpI 1 escape X inactivation (Miller et al. 1995). As the density of markers across the chromosome has increased beyond the 100-kb resolution goal suggested for the 1Corresponding author. E-MAIL davids@sequencer.wustl.edu; FAX (314) 362-3203."genome initiative," additional features are revealed, as described here.The average inter-STS distance of-75 kb has been achieved by the placement of 2091 STSs on cognate YACs across the 160 Mb of the chromosome. Collectively, the STSs sample -1% of Xspecific sequences. About half of the STSs (962) are made from YAC insert ends (Kere et al. 1992), and another 592 are from randomly derived unique Xchromosomal sequences. However, the STSs also include 97 expressed sequence tags (ESTs) and 190 gene-specific STSs from known genes, as well as 192 dinucleotide and 38 tri-and tetranucleotide repeat markers that detect polymorphism. As a result, the YAC/STS map can be integrated with transcriptional and genetic maps. RESULTS Mapping Strategy and PerformanceWe used a modified "all-walking" form of STS content mapping (Kere et al. 1992) in which STSs were

A protein which initiates assembly of ribosomes is defined as a protein which binds to the respective rRNA without cooperativity (i.e., without the help of other proteins) during the onset of assembly and is essential for the formation of active ribosomal subunits. The number of proteins binding without cooperativity was determined by monitoring the reconstitution output of active particles at various inputs of 16S rRNA, in the presence of constant amounts of 30S-derived proteins (TP30): This showed that only two of the proteins of the 30S subunit are assembly-initiator proteins. These two proteins are still present on a LiCl core particle comprising 16S rRNA and 12 proteins (including minor proteins). The 12 proteins were isolated, and a series of reconstitution experiments at various levels of rRNA excess demonstrated that S4 and S7 are the initiator proteins. Pulse-chase experiments performed during the early assembly with 14C- and 3H-labeled TP30 and the determination of the 14C/3H ratio of the individual proteins within the assembled particles revealed a bilobal structure of the 30S assembly: A group of six proteins headed by S4 (namely, S4, S20, S16, S15, S6, and S18) resisted the chasing most efficiently (S4 assembly domain). None of the proteins depending on S7 during assembly were found in this group but rather in a second group with intermediate chasing stability [S7 assembly domain; consisting of S7, S9, (S8), S19, and S3]. A number of proteins could be fully chased during the early assembly and therefore represent "late assembly proteins" (S10, S5, S13, S2, S21, S1). These findings fit well with the 30S assembly map.(ABSTRACT TRUNCATED AT 250 WORDS)

An rRNA-binding protein that binds to the rRNA independently of other proteins during the course of ribosomal assembly is termed "assembly initiator protein." In spite of the large number of rRNA-binding proteins (more than 17 out of 32 proteins have been identified in the case of the large ribosomal subunit), only a very small number of proteins should actually initiate ribosomal assembly for theoretical reasons. Here we demonstrate that only two of the L proteins derived from the large subunit (50S) function as assembly initiator proteins. Two different techniques are used to identify these initiator proteins: reconstitution experiments with purified proteins and pulse-chase experiments during in vitro assembly. Both methods independently identify L24 and L3 as initiator proteins for the 50S assembly. The existence of two initiator proteins (not just one) resolves an apparent contradiction-namely, that on the one hand, rRNA is synthesized in excess under unfavorable growth conditions, whereas on the other hand, rRNA-binding proteins should be available for translational control.The majority ofthe ribosomal proteins in both the small and the large ribosomal subunit bind in vitro to their respective rRNA. In the small subunit, 12 out of21 proteins have been identified as "RNA-binding proteins" (1,2), and in the large subunit, 17 out of 32 (3, 4). Proteins derived from the large ribosomal subunit are designated L.In the case ofthe small ribosomal subunit, it has been shown that the cooperativity of the assembly process is so high that certainly not all 12 but at most 2 or 3 proteins (or protein complexes) can bind independently to 16S rRNA (5).In the case of the large (50S) subunit, it is not yet clear whether even the seven most strongly binding proteins (4) bind independently to their rRNA in the course of the assembly. If this were so, then these proteins would be distributed independently of each other over the various individual rRNA molecules. If the rRNA were present in excess, this random distribution would disturb the formation of ribosomal subunits with a constant and stoichiometric protein content and, as a result, dramatically decrease the output ofactive ribosomes. In fact, a synthesis of rRNA in a molar excess of more than threefold over ribosomal proteinst has been observed under unfavorable growth conditions (6). Therefore, one would expect that, in spite ofthe large number of23S rRNA-binding proteins concerned in the assembly ofthe 50S subunit, only a few ofthese proteins should be able to bind independently to the rRNA (i. e., without cooperativity), thus selecting the rRNA molecules for the 50S assembly in the presence of an rRNA excess. These proteins we term "assembly initiator proteins".In this paper we demonstrate that the in vitro assembly of the large ribosomal subunit is initiated by only two proteins.Two different methods independently identify these proteins as L24 and L3. MATERIAL AND METHODSRibosomes and their subunits were isolated from Escherichia coli cells as described (...

The release of amyloid-beta peptide (Abeta) from beta-amyloid precursor protein (APP) requires cleavage by beta- and gamma-secretases. Several groups have identified a candidate for the beta-site APP-cleaving enzyme, BACE1, and its homologue BACE2. We sequenced the genes for BACE1 and BACE2 and found several polymorphisms in both genes. Genotyping a large cohort of AD cases and controls has shown no association between AD and the intronic polymorphism in BACE2 while there was a weak association between the BACE1 polymorphism in exon 5 and AD in those carrying the APOE epsilon4 allele.

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