Mutagenesis by third-strand-directed psoralen adducts in repair-deficient human cells: high frequency and altered spectrum in a xeroderma pigmentosum variant.

Psoralen-conjugated triple-helix-forming oligonucleotides have been used to generate site-specific mutations within mammalian cells. To investigate factors influencing the efficiency of oligonucleotide-mediated gene targeting, the processing of third-strand-directed psoralen adducts was compared in normal and repair-deficient human cells. An unusually high mutation frequency and an altered mutation pattern were seen in xeroderma pigmentosum variant (XPV) cells compared with normal, xeroderma pigmentosum group A (XPA), and Fanconi anemia cells. In XPV, targeted mutations were produced in the supF reporter gene carried in a simian virus 40 vector at a frequency of 30%, 3-fold above that in normal or Fanconi anemia cells and 6-fold above that in XPA. The mutations generated by targeted psoralen crosslinks and monoadducts in the XPV cells formed a pattern distinct from that in the other three cell lines, with mutations occurring not just at the damaged site but also at adjacent base pairs. Hence, the XPV cells may have an abnormality in trans-lesion bypass synthesis during repair and/or replication, implicating a DNA polymerase or an accessory factor as a basis of the defect in XPV. These results may help to elucidate the repair deficiency in XPV, and they raise the possibility that genetic manipulation via triplex-targeted mutagenesis may be enhanced by modulation of the XPV-associated activity in normal cells.

Myristate exchange on the Trypanosoma brucei variant surface glycoprotein.

The glycosyl-phosphatidylinositol (GPI) anchor of the Trypanosoma brucei variant surface glycoprotein (VSG) is unique in having exclusively myristate as its fatty acid component. We previously demonstrated that the myristate specificity is the result of two independent pathways. First, the newly synthesized free GPI, which is not myristoylated, undergoes fatty acid remodeling to replace both its fatty acids with myristate. Second, the myristoylated precursor, glycolipid A, undergoes a myristate exchange reaction, detected by the replacement of unlabeled myristate by [3H]myristate. Remodeling and exchange have different enzymatic properties and apparently occur in different subcellular compartments. We now demonstrate that the GPI anchor linked to VSG is the major substrate for myristate exchange. VSG can be efficiently labeled with [3H]myristate by exchange in the presence of cycloheximide, an inhibitor that prevents new VSG synthesis and thus anchor addition to protein. Not only is newly synthesized VSG subject to exchange, but mature VSG, possibly recycling from the cell surface, also undergoes myristate exchange.

Vascular variant of prion protein cerebral amyloidosis with tau-positive neurofibrillary tangles: the phenotype of the stop codon 145 mutation in PRNP.

Deposition of PrP amyloid in cerebral vessels in conjunction with neurofibrillary lesions is the neuropathologic hallmark of the dementia associated with a stop mutation at codon 145 of PRNP, the gene encoding the prion protein (PrP). In this disorder, the vascular amyloid in tissue sections and the approximately 7.5-kDa fragment extracted from amyloid are labeled by antibodies to epitopes located in the PrP sequence including amino acids 90-147. Amyloid-laden vessels are also labeled by antibodies against the C terminus, suggesting that PrP from the normal allele is involved in the pathologic process. Abundant neurofibrillary lesions are present in the cerebral gray matter. They are composed of paired helical filaments, are labeled with antibodies that recognize multiple phosphorylation sites in tau protein, and are similar to those observed in Alzheimer disease. A PrP cerebral amyloid angiopathy has not been reported in diseases caused by PRNP mutations or in human transmissible spongiform encephalopathies; we propose to name this phenotype PrP cerebral amyloid angiopathy (PrP-CAA).

Improved biodistribution, tumor targeting, and reduced immunogenicity in mice with a gamma 4 variant of Campath-1H.

Radiolabeled antibodies have shown promise for the treatment of lymphoma and for solid tumor targeting. Campath-1H is a humanized monoclonal antibody that reacts with the CD52 antigen present on human lymphoid and myeloid cells. Campath-1H is a gamma1 (G1) isotype that induces lymphopenia via an Fc-mediated mechanism(s). Isotype switches were engineered, and the resulting antibodies were expressed in NS0 mouse myeloma cells and biosynthetically radiolabeled with [35S]methionine. The forms included G1, G4, and a G4 variant that contained alanine substitutions at (EU numbering) Leu-235, Gly-237, and Glu-318. All isotypes bound antigen equivalently as assessed by target cell binding in vitro. The G4 variant had a greatly reduced capacity to interact with Fc receptor by virtue of reduced binding to THP-1 human myeloid cells and by a 1000-fold increase in EC50 to intermediate antibody-dependent cellular cytotoxicity. The pharmacokinetics of the isotypes were compared in CD-1 (nu/nu) mice bearing an experimental antigen-expressing tumor. The plasma half-life and tumor uptake were increased for the G4 variant. The G4 variant showed significantly less spleen, liver, and bone uptake but similar uptake in the lung, kidney, and stomach and lower tissue-to-blood ratios. Immunogenicity was assessed after repeated monthly administrations of unlabeled antibody in BALB/c mice. A 50% reduction in the incidence of anti-globulin response was observed for the G4 variant. These properties suggest that antibodies with reduced Fc receptor interaction merit additional study as potential targeting vehicles relative to other isotypes for radioimmunotherapy or situations where diminished normal tissue binding contributes to efficacy.

Assembly of regularly spaced nucleosome arrays by Drosophila chromatin assembly factor 1 and a 56-kDa histone-binding protein.

To ascertain the mechanism by which nucleosomes are assembled by factors derived from Drosophila embryos, two proteins termed Drosophila chromatin assembly factors (CAFs) 1 and 4 (dCAF-1 and dCAF-4) were fractionated and purified from a Drosophila embryo extract. The assembly of chromatin by dCAF-1, dCAF-4, purified histones, ATP, and DNA is a process that generates regularly spaced nucleosomal arrays with a repeat length that resembles that of bulk native Drosophila chromatin and is not obligatorily coupled to DNA replication. The assembly of chromatin by dCAF-1 and dCAF-4 is nearly complete within 10 min. The dCAF-1 activity copurified with the Drosophila version of chromatin assembly factor-1 (CAF-1), a factor that has been found to be required for the assembly of chromatin during large tumor (T) antigen-mediated, simian virus 40 (SV40) origin-dependent DNA replication. The dCAF-4 activity copurified with a 56-kDa core-histone-binding protein that was purified to > 90% homogeneity.

The histone fold: a ubiquitous architectural motif utilized in DNA compaction and protein dimerization.

The histones of all eukaryotes show only a low degree of primary structure homology, but our earlier crystallographic results defined a three-dimensional structural motif, the histone fold, common to all core histones. We now examine the specific architectural patterns within the fold and analyze the nature of the amino acid residues within its functional segments. The histone fold emerges as a fundamental protein dimerization motif while the differentiations of the tips of the histone dimers appear to provide the rules of core octamer assembly and the basis for nucleosome regulation. We present evidence for the occurrence of the fold from archaebacteria to mammals and propose the use of this structural motif to define a distinct family of proteins, the histone fold superfamily. It appears that evolution has conserved the conformation of the fold even through variations in primary structure and among proteins with various functional roles.

Cell cycle regulation of the cyclin A gene promoter is mediated by a variant E2F site.

Cyclin A is involved in the control of S phase and mitosis in mammalian cells. Expression of the cyclin A gene in nontransformed cells is characterized by repression of its promoter during the G1 phase of the cell cycle and its induction at S-phase entry. We show that this mode of regulation is mediated by the transcription factor E2F, which binds to a specific site in the cyclin A promoter. It differs from the prototype E2F site in nucleotide sequence and protein binding; it is bound by E2F complexes containing cyclin E and p107 but not pRB. Ectopic expression of cyclin D1 triggers premature activation of the cyclin A promoter by E2F, and this effect is blocked by the tumor suppressor protein p16INK4.

A variant of lambda repressor with an altered pattern of cooperative binding to DNA sites.

The bacteriophage lambda repressor binds cooperatively to pairs of adjacent sites in the lambda chromosome, one repressor dimer binding to each site. The repressor's amino domain (that which mediates DNA binding) is connected to its carboxyl domain (that which mediates dimerization and the interaction between dimers) by a protease-sensitive linker region. We have generated a variant lambda repressor that lacks this linker region. We show that dimers of the variant protein are deficient in cooperative binding to sites at certain, but not all, distances. The linker region thus extends the range over which carboxyl domains of DNA-bound dimers can interact. In particular, the linker is required for cooperative binding to a pair of sites as found in the lambda chromosome, and thus is essential for the repressor's physiological function.

Restoration of surface IgM-mediated apoptosis in an anti-IgM-resistant variant of WEHI-231 lymphoma cells by HS1, a protein-tyrosine kinase substrate.

The HS1 protein is one of the major substrates of non-receptor-type protein-tyrosine kinases and is phosphorylated immediately after crosslinking of the surface IgM on B cells. The mouse B-lymphoma cell line WEHI-231 is known to undergo apoptosis upon crosslinking of surface IgM by anti-IgM antibodies. Variants of WEHI-231 that were resistant to anti-IgM-induced apoptosis expressed dramatically reduced levels of HS1 protein. Expression of the human HS1 protein from an expression vector introduced into one of the variant cell lines restored the sensitivity of the cells to apoptosis induced by surface IgM crosslinking. These results suggest that HS1 protein plays a crucial role in the B-cell antigen receptor-mediated signal transduction pathway that leads to apoptosis.

A splice variant of alpha 6 integrin is associated with malignant conversion in mouse skin tumorigenesis.

The epithelial-specific integrin alpha 6 beta 4 is suprabasally expressed in benign skin tumors (papillomas) and is diffusely expressed in carcinomas associated with an increase in the proliferating compartment. Analysis of RNA samples by reverse transcriptase-PCR and DNA sequencing revealed that chemically or oncogenically induced papillomas (n = 8) expressed a single transcript of the alpha 6 subunit, identified as the alpha 6 A splice variant. In contrast, carcinomas (n = 13) expressed both alpha 6A and an alternatively spliced form, alpha 6B. Primary keratinocytes and a number of keratinocyte cell lines that vary in biological potential from normal skin, to benign papillomas, to well-differentiated slowly growing carcinomas exclusively expressed alpha 6A. However, I7, an oncogene-induced cell line that produces highly invasive carcinomas, expressed both alpha 6A and alpha 6B transcript and protein. The expression of alpha 6B in I7 cells was associated with increased attachment to a laminin matrix compared to cell lines exclusively expressing alpha 6A. Furthermore, introduction of an alpha 6B expression vector into a papilloma cell line expressing alpha 6A increased laminin attachment. When a papilloma cell line was converted to an invasive carcinoma by introduction of the v-fos oncogene, the malignant cells expressed both alpha 6A and alpha 6B, while the parent cell line and cells transduced with v-jun or c-myc, which retained the papilloma phenotype, expressed only alpha 6A. Comparative analysis of alpha 6B expression in cell lines and their derived tumors indicate that alpha 6B transcripts are more abundant in tumors than cell lines, and alpha 6B is expressed to a greater extent in poorly differentiated tumors. These results establish a link between malignant conversion and invasion of squamous tumor cells and the regulation of transcript processing of the alpha 6 beta 4 integrin.

High-affinity binding sites for histone H1 in plasmid DNA.

The interaction of histone H1 isolated from chicken erythrocytes with restriction fragments from plasmids pBR322 and pUC19 was studied by gel electrophoresis. Certain restriction fragments exhibited unusually high affinity for the histone, forming high molecular mass complexes at protein to DNA ratios at which the other fragments did not show evidence for binding. The highly preferred fragments are intrinsically curved, as judged by their electrophoretic mobility in polyacrylamide gels, by computer modeling, and by imaging with scanning force microscopy. However, control experiments with either curved portions of the same fragments or highly curved kinetoplast DNA fragments showed that the presence of curvature alone was not sufficient for preferential binding. By using various restriction fragments centered around the highly preferred sequence, it was found that the high-affinity binding required in addition the presence of specific sequences on both sides of the region of curvature. Thus, both curvature and the presence of specific sites seem to be required to generate high affinity.

An activity gel assay detects a single, catalytically active histone acetyltransferase subunit in Tetrahymena macronuclei.

Macronuclei of the ciliated protozoan Tetrahymena thermophila possess a histone acetyltransferase activity closely associated with transcription-related histone acetylation. Nothing definitive is known concerning the polypeptide composition of this activity in Tetrahymena or any comparable activity from any cellular source. An acetyltransferase activity gel assay was developed which identifies a catalytically active subunit of this enzyme in Tetrahymena. This activity gel assay detects a single polypeptide of 55 kDa (p55) in crude macronuclear extracts, as well as in column-purified fractions, which incorporates [3H]acetate from [3H]acetyl-CoA into core histone substrates polymerized directly into SDS polyacrylamide gels. p55 copurifies precisely with acetyltransferase activity through all chromatographic steps examined, including reverse-phase HPLC. Gel-filtration chromatography of this activity indicates a molecular mass of 220 kDa, suggesting that the native enzyme may consist of four identical subunits of 55 kDa. Furthermore, p55 is tightly associated with di- and greater polynucleosomes and therefore may be defined as a component of histone acetyltransferase type A--i.e., chromatin associated.

Yeast histone H3 and H4 N termini function through different GAL1 regulatory elements to repress and activate transcription.

Previous work has shown that N-terminal deletions of yeast histone H3 cause a 2- to 4-fold increase in the induction of GAL1 and a number of other genes involved in galactose metabolism. In contrast, deletions at the H4 N terminus cause a 10- to 20-fold decrease in the induction of these same GAL genes. However, H3 and H4 N-terminal deletions each decrease PHO5 induction only 2- to 4-fold. To define the GAL1 gene regulatory elements through which the histone N termini activate or repress transcription, fusions were made between GAL1 and PHO5 promoter elements attached to a beta-galactosidase reporter gene. We show here that GAL1 hyperactivation caused by the H3 N-terminal deletion delta 4-15 is linked to the upstream activation sequence. Conversely, the relative decrease in GAL1 induction caused by the H4N-terminal deletion delta 4-28 is linked to the downstream promoter which contains the TATA element. These data indicate that the H3 N terminus is required for the repression of the GAL1 upstream element, whereas the H4N terminus is required for the activation of the GAL1 downstream promoter element.

Insulin-like growth factor I receptors of fetal brain are enriched in nerve growth cones and contain a beta-subunit variant.

Nerve growth cones isolated from fetal rat brain are highly enriched in a 97-kDa glycoprotein, termed beta gc, that comigrates with the beta subunit of the IGF-I receptor upon two-dimensional PAGE and is disulfide-linked to this receptor's alpha subunit. Antibodies prepared to a conserved domain shared by the insulin and IGF-I receptor beta subunits (AbP2) or to beta gc were used to study receptor distribution further. Subcellular fractionation of the fetal brain segregated most AbP2 immunoreactivity away from growth cones, whereas most beta gc immunoreactivity copurified with growth cones. Experiments involving ligand-activated receptor autophosphorylation confirmed the concentration of IGF-I but not of insulin receptors in growth cone fractions. These results indicate the enrichment of IGF-I receptors in (presumably axonal) growth cones of the differentiating neuron. Furthermore, the segregation of beta gc from AbP2 immunoreactivity suggests that such neurons express an immunochemically distinct variant of the IGF-I receptor beta subunit at the growth cone.