Robert F. Clark

A mutation in Alzheimer's disease destroying a splice acceptor site in the presenilin-1 gene

A series of mutations has been reported in the presenilin-1 (PS-1) gene which cause early onset Alzheimers disease (AD). The mutations reported to date have encoded missense mutations which alter residues conserved between PS-1 and the presenilin-2 (PS-2) gene. We have recently determined the intron/exon structure of the PS-1 gene and this information has been used to identify a mutation in the splice acceptor site for exon 9 in a family with early onset AD. Amplification of cDNA from lymphoblasts of affected individuals revealed that the effect of the mutation was to cause splicing out of exon 9, however it does not change the open reading frame of the mRNA. The importance of this observation is discussed.

Complete analysis of the presenilin 1 gene in early onset Alzheimer's disease

The presenilin 1 gene has recently been identified as the locus on chromosome 14 which is responsible for a large proportion of early onset, autosomal dominantly inherited Alzheimers disease (AD). We have elucidated the intron/exon structure of the gene and designed intronic primers to enable direct sequencing of the entire coding region (10 exons) of the presenilin gene in a large number of families. This strategy has enabled us to find a further two novel mutations in the gene. We discuss the distribution of mutations and the proportions of autosomal dominant AD with a mean age of onset below 60 years caused by mutations in this gene.

Structure and alternative splicing of the presenilin-2 gene.

Missense mutations in the presenilin-1 (PS-1) and presenilin-2 (PS-2) genes have been shown to be causes of autosomal dominant Alzheimers disease (the AD3 and AD4 loci, respectively). Alternative splicing has previously been reported in the PS-1 gene. In this study, elucidation of intron/exon boundary sequences revealed that PS-2 is encoded by 10 coding exons. In addition, PS-2 cDNA cloning and RT-PCR using RNA from a variety of normal tissues revealed the presence of alternatively spliced products. These products included species with in frame omissions of exon 8 and simultaneous omissions of exons 3 and 4.

Fractionation of human H1 subtypes and characterization of a subtype-specific antibody exhibiting non-uniform nuclear staining

Four histone H1 subtypes and H1° were fractionated from human placental nuclei and purified to homogeneity by a combination of Bio-Rex 70 chromatography and reversephase high-performance liquid chromatography (RPHPLC). Polyclonal antibodies were generated in rabbits against one of these subtypes designated H1-3. Antibodies reacted only against this subtype in enzymelinked immunosorbent assays and Western assays; subtype specificity was documented further by Western blotting of cell and nuclear extracts. They crossreacted with monkey H1, but not with H1 from other vertebrates tested. The epitope(s) recognized were mapped by immunoblotting against peptides prepared by cleavage with N-bromosuccinimide (NBS) and α-chymotrypsin; it includes the variant amino-terminal tail of the protein as well as a portion of the globular domain. The antibody stains mitotic chromosomes weakly but uniformly and, unlike antibodies that recognize total H1 which show uniform nuclear staining after indirect immunofluoresence localization, anti-H1-3 exhibits preferential labelling of the nuclear periphery. This non-uniform staining suggests compartmentalization of this subtype which may have functional significance with respect to differential chromatin condensation.

Posttranslational modification and plasma membrane localization of the Drosophila melanogaster presenilin.

Mutations in two genes, presenilin 1 (PS1) and presenilin 2, are linked to early onset cases of familial Alzheimers disease. The presenilins are thought to contribute to the pathogenesis of Alzheimers disease by directly or indirectly affecting the proteolytic processing of the amyloid precursor protein. They have also been implicated in the proteolytic processing of Notch. In PS1-deficient mammalian cells, the proteolytic release of the Notch intracellular domain is reduced. Likewise, loss-of-function mutations in Drosophila presenilin (Psn) prevent the production of the intracellular Notch signaling fragment and lead to phenotypes resembling Notch mutants. Here we characterize the Drosophila Psn protein and demonstrate that it undergoes a proteolytic cleavage. We describe Psn expression at different developmental stages of the fly and show Psn localization near both apical and basal plasma membranes. Furthermore, we demonstrate that portions of the Psn protein span the plasma membrane in S2 cells.

THE ROLE OF PRESENILIN 1 IN THE GENETICS OF ALZHEIMER'S DISEASE

Approximately 75% of AD patients have an onset of the disease after the age of 60 years, and 60% of AD patients have no family history of the disease. Some cases of EOAD are clearly inherited in an autosomal-dominant manner. The beta APP gene on chromosome 21, the PS-1 gene on chromosome 14, and the PS-2 gene on chromosome 1 have all been characterized as genes in which mutations lead to familial EOAD. For LOAD, the work on ApoE indicates that the epsilon 4 allele is a risk factor for developing AD. However, 35-50% of all AD patients do not have an epsilon 4 allele. Other loci contributing to LOAD remain to be mapped and characterized. As in other complex disorders, these additional loci may involve genetic interactions with the known AD loci. Identification of all susceptibility loci for AD is a major goal in resolving the pathogenesis of AD.

Chapter 8 Distribution of Chromosomal Proteins in Polytene Chromosomes of Drosophila

Publisher Summary This chapter discusses the distribution of chromosomal proteins in polytene chromosomes of Drosophila . The gene regulation is facilitated by the identification and characterization of proteins that interact with DNA. The chapter identifies and characterizes proteins specifically associated with either euchromatin or heterochromatin. Techniques that allow establishing the pattern of interaction between a given protein and the genome as a whole are used. In Drosophila , many of the chromosomal proteins are studied by using the technique described in the chapter, that of immunolocalization of proteins on polytene chromosomes. The chapter presents a technique for determining the in situ distribution of chromosomal proteins in polytene chromosomes with the emphasis on a procedure using formaldehyde fixation followed by squashing in acetic acid. The chapter also discusses the characteristics for ideal polytene chromosomes for immunofluorescent microscopy. To localize the positions of chromosomal proteins on the polytene chromosomes, the chromosome spreads are reacted with a primary antibody against a particular protein, followed by reaction with a fluorescent or other secondary antibody against the primary antibody.