Gary E. Dean

Cloning and functional expression of a tetrabenazine sensitive vesicular monoamine transporter from bovine chromaffin granules

Using oligonucleotide primers derived from the vesicular monoamine transporters sequences, a cDNA predicted to encode the bovine chromaffin granule amine transporter has been cloned (b‐VMAT2). Surprisingly, its structure is more similar to the rat brain transporter (VMAT2), than to the rat adrenal counterpart (VMAT1). Unlike rat VMAT1, bovine VMAT2 appears to be expressed both in the adrenal medulla and the brain, as judged by Northern analysis. After modification/deletion of the seven amino acids at the N‐terminus of the protein it was expressed in a functional form. The order of affinity of the bovine VMAT2 transporter to substrates is: serotonin>dopamine = norepinephrine>epinephrine. Also, the recombinant bovine adrenal transporter is highly sensitive to tetrabenazine, in sharp contrast to the rat adrenal transporter. The findings indicate, therefore, a clear species variation in which structure and function of the bovine adrenal transporter resemble the rat brain protein, while its tissue distribution is distinct from both types of rat proteins. In addition, the predicted protein sequence is identical to the experimentally determined N‐terminus sequence of the purified vesicular amine transporter [Stern‐Bach et al. (1992) Proc. Natl. Acad. Sci. USA 89, 9730‐9733].

AnkB, a Periplasmic Ankyrin-Like Protein in Pseudomonas aeruginosa, Is Required for Optimal Catalase B (KatB) Activity and Resistance to Hydrogen Peroxide

In this study, we have cloned the ankB gene, encoding an ankyrin-like protein in Pseudomonas aeruginosa. The ankB gene is composed of 549 bp encoding a protein of 183 amino acids that possesses four 33-amino-acid ankyrin repeats that are a hallmark of erythrocyte and brain ankyrins. The location of ankB is 57 bp downstream of katB, encoding a hydrogen peroxide-inducible catalase, KatB. Monomeric AnkB is a 19.4-kDa protein with a pI of 5.5 that possesses 22 primarily hydrophobic amino acids at residues 3 to 25, predicting an inner-membrane-spanning motif with the N terminus in the cytoplasm and the C terminus in the periplasm. Such an orientation in the cytoplasmic membrane and, ultimately, periplasmic space was confirmed using AnkB-BlaM and AnkB-PhoA protein fusions. Circular dichroism analysis of recombinant AnkB minus its signal peptide revealed a secondary structure that is approximately 65% alpha-helical. RNase protection and KatB- and AnkB-LacZ translational fusion analyses indicated that katB and ankB are part of a small operon whose transcription is induced dramatically by H(2)O(2), and controlled by the global transactivator OxyR. Interestingly, unlike the spherical nature of ankyrin-deficient erythrocytes, the cellular morphology of an ankB mutant was identical to that of wild-type bacteria, yet the mutant produced more membrane vesicles. The mutant also exhibited a fourfold reduction in KatB activity and increased sensitivity to H(2)O(2), phenotypes that could be complemented in trans by a plasmid constitutively expressing ankB. Our results suggest that AnkB may form an antioxidant scaffolding with KatB in the periplasm at the cytoplasmic membrane, thus providing a protective lattice work for optimal H(2)O(2) detoxification.

A new method to remove hybridization bias for interspecies comparison of global gene expression profiles uncovers an association between mRNA sequence divergence and differential gene expression in Xenopus

The recent sequencing of a large number of Xenopus tropicalis expressed sequences has allowed development of a high-throughput approach to study Xenopus global RNA gene expression. We examined the global gene expression similarities and differences between the historically significant Xenopus laevis model system and the increasingly used X.tropicalis model system and assessed whether an X.tropicalis microarray platform can be used for X.laevis. These closely related species were also used to investigate a more general question: is there an association between mRNA sequence divergence and differences in gene expression levels? We carried out a comprehensive comparison of global gene expression profiles using microarrays of different tissues and developmental stages of X.laevis and X.tropicalis. We (i) show that the X.tropicalis probes provide an efficacious microarray platform for X.laevis, (ii) describe methods to compare interspecies mRNA profiles that correct differences in hybridization efficiency and (iii) show independently of hybridization bias that as mRNA sequence divergence increases between X.laevis and X.tropicalis differences in mRNA expression levels also increase.

Genetic Literacy of Undergraduate Non–Science Majors and the Impact of Introductory Biology and Genetics Courses

ABSTRACT With the advancement of genetic information and technologies, there is an increasing need for a genetically literate public. This study looks critically at student learning and at the current instruction of genetics in introductory non–science major biology and genetics courses at the undergraduate level. A new diagnostic tool, the Genetic Literacy Assessment Instrument, was administered pre- and postcourse to more than 300 students in six introductory nonmajor courses that emphasize genetics to varying degrees. Current data from students in these courses show a precourse average score of 43 percent correct zn the inventory. Postcourse scores increased only modestly, to an average of 49 percent. In this article, we discuss the impact of teaching methods and course content on scores, as well as student learning in the different content areas of genetics. The results suggest that further studies in genetics education are needed to better understand the effect of teaching methods on achieving genetic literacy.

A PC12 variant lacking regulated secretory organelles: aberrant protein targeting and evidence for a factor inhibiting neuroendocrine gene expression.

Abstract: A variant of the PC12 pheochromocytoma cell line (termed A35C) has been isolated that lacks regulated secretory organelles and several constituent proteins. Northern and Southern blot analyses suggested a block at the transcriptional level. The proprotein‐converting enzyme carboxypeptidase H was synthesised in the A35C cell line but was secreted by the constitutive pathway. Transient transfection of A35C cells with cDNAs encoding the regulated secretory proteins dopamine β‐hydroxylase and synaptotagmin I resulted in distinct patterns of mistargeting of these proteins. It is surprising that hybrid cells created by fusing normal PC12 cells with A35C cells exhibited the variant phenotype, suggesting that A35C cells express an inhibitory factor that represses neuroendocrine‐specific gene expression.

Structure and expression of subunit A from the bovine chromaffin cell vacuolar ATPase

Subunit A of the vacuolar H+‐ATPase class is thought to be responsible for the ATP hydrolysis which drives proton‐pumping. We report here the cloning and sequence determination of the first mammalian cDNA encoding a bovine vacuolar ATPase subunit A from an adrenal medulla cDNA library. Northern blots of bovine adrenal medulla RNA reveal a message of approximately 3.8 kb. The predicted peptide sequence, consisting of 618 amino acids with a calculated molecular weight of 68397 daltons, is similar to the sequences of the three known subunit A proteins. β‐Galactosidase‐subunit A fusion proteins were immuno‐decorated by an antiserum raised to the subunit A protein from corn coleoptile vacuoles.

Cloning and sequence analysis of an H+-ATPase-encoding gene from the human dimorphic pathogen Histoplasma capsulatum

A gene related to the PMA1 gene from Saccharomyces cerevisiae was isolated from the pathogenic human dimorphic fungus, Histoplasma capsulatum, using fungal-specific oligodeoxyribonucleotide (oligo) probes. This gene has been given the name Hc-PMA1. The structural organization of Hc-PMA1 consists of three exons (375, 2329 and 44 bp) and two introns (115 and 116 bp). The nucleotide sequence predicts an H(+)-ATPase-related protein of 916 amino acids (aa). Comparison of the deduced aa sequence to that of Neurospora crassa and S. cerevisiae (PMA1) plasma membrane H(+)-ATPases showed a greater similarity to that from N. crassa (85% identity). Furthermore, the two introns in the Hc-PMA1 gene interrupt the coding region in the precise locations determined for two of the four N. crassa Nc-PMA introns. H. capsulatum intron 1 contains two repeat motifs, d(TA)16 and d(TG)10, each potentially capable of forming non-B DNA structures. Northern analysis of H. capsulatum total RNA indicated that the Hc-PMA1-specific mRNA is approx. 3.3 kb in size, in agreement with the predicted size of the gene.

Purification and Solubilization of Paired Helical Filaments from Alzheimer Brains

Abstract: The purpose of the present study was to develop a purification and solubilization method, compatible with current amino acid sequencing techniques, for paired helical filaments (PHFs) derived from patients with Alzheimers disease. We have developed a mild procedure that subjects conventionally isolated PHFs to Tris/borate/sodium dodecyl sulfate/2‐mercaptoethanol electrophoresis and results in the separation of the relatively insoluble PHF structures from both copurifying contaminating proteins and solubilized PHF‐associated proteins. At the end of 4.5 h of electrophoresis, the purified insoluble fraction had an amino acid composition that was invariant during subsequent electrophoresis. Electron microscopy revealed an intact PHF structure before and after electrophoresis but no evidence of any other structures in the insoluble fraction, a result consistent with the removal of PHF‐associated proteins from the filament structure. Isolated insoluble filament structures displayed an enhanced immunoreactivity with antibodies raised against purified PHFs in other laboratories, when compared with the fraction not subjected to electrophoresis in enzyme‐linked immunosorbent assays. Solubilization of the relatively insoluble PHFs was accomplished by extending the time of electrophoresis beyond the 4.5 h required for purification. Additional electrophoresis for 34.5 h solubilized 88% of the purified, relatively insoluble PHFs. This resulted in the identification of four major protein bands between Mr values of ∼50,000 and 70,000 on sodium dodecyl sulfate‐polyacrylamide electrophoresis gel analysis, with a predominant band with an Mr of ∼66,000. A slow fragmentation of the PHF ultrastructure occurred during this time, as judged by electron microscopy. This purification technique will permit the isolation of consistently reproducible protein fragments from solubilized PHFs, which may be used for subsequent sequence analysis.

MONOCLONAL ANTIBODY PHF-9 RECOGNIZES PHOSPHORYLATED SERINE 404 OF TAU PROTEIN AND LABELS PAIRED HELICAL FILAMENTS

Paired helical filaments (PHFs) purified from Alzheimers brain consist of hyperphosphorylated microtubule‐associated protein tau. In PHF, phosphorylation occurs at ser/thr tau residues. Several of these ser/thr phosphorylation sites lie immediately C‐terminal to the tau tubulin binding domain. The C‐terminal ser396 to thr413 tau region contains two or more phosphorylated residues and eight possible ser/thr phosphorylation sites. Immunologic studies and mass spectroscopy have identified ser396 as one of the phosphorylation sites but identification of more C‐terminal phosphorylated residues has been hampered by the lack of monoclonal antibodies (Mabs) that recognize defined epitopes in this region. We have raised Mabs against PHF purified from Alzheimers brain. One of these Mabs, PHF‐9, showed phosphorylation‐dependent binding to purified PHF and recognized a phosphorylated epitope in the C‐terminal portion of cyanogen bromide‐digested PHF. Epitope mapping studies employing synthetic tau phosphopeptides indicated that PHF‐9 labeled a 13‐mer tau peptide phosphorylated at ser404 but not the corresponding non‐phosphorylated peptide. PHF‐9 demonstrated no immunoreactivity with a synthetic peptide phosphorylated at ser396 indicating that the PHF‐9 epitope is C‐terminal to ser396. In conclusion, the present study describes a Mab, PHF‐9, which recognizes phosphorylated ser404 of tau independently of phosphorylated ser396 and indicates that tau ser404 is phosphorylated in PHF.

Identification of microtubule-associated protein tau isoforms in Alzheimer's paired helical filaments.

AD66 proteins derived from sodium dodecylsulfate (SDS) insoluble paired helical filaments (PHF) were isolated from Alzheimers brain using a purification procedure developed previously in this laboratory, and characterized by immunologic and chemical cleavage methods. AD66 proteins were immunoreactive with antibodies that recognize the amino terminal, tubulin-binding, and carboxy terminal domains of microtubule-associated protein tau indicating the presence of the entire tau sequence in AD66 proteins. These proteins were reactive with antibody 423 that binds to PHF but not human adult tau. Immunologic and chemical cleavage studies indicated that only two of the six tau isoforms were present in these proteins. AD66 proteins were comprised of tau proteins containing only three tubulin binding domains with either a 29 amino acid insert or no amino terminal insert. For comparative purposes, SDS soluble PHF-tau (A68 proteins) was purified from Alzheimers brains and normal adult tau purified from control brains. Antibody Alz-50 was immunoreactive with PHF-tau or normal tau regardless of alkaline phosphatase treatment while immunoreactivity was only observed with dephosphorylated AD66 proteins. A second phosphorylated epitope on AD66 proteins but not PHF-tau or normal tau proteins was demonstrated with antibody PHF9. These data suggest that AD66 proteins represent a more phosphorylated form of tau than PHF-tau or normal tau proteins. Two-dimensional gel electrophoresis demonstrated that AD66 proteins have higher apparent molecular weights and lower pI values than normal tau, differences possibly due to the greater phosphorylation observed in these proteins.