Andrew W. Dangel

The gene mutated in bare patches and striated mice encodes a novel 3β-hydroxysteroid dehydrogenase

X-linked dominant disorders that are exclusively lethal prenatally in hemizygous males have been described in human and mouse. None of the genes responsible has been isolated in either species. The bare patches (Bpa ) and striated (Str) mouse mutations were originally identified in female offspring of X-irradiated males. Subsequently, additional independent alleles were described. We have previously mapped these X-linked dominant, male-lethal mutations to an overlapping region of 600 kb that is homologous to human Xq28 (ref. 4) and identified several candidate genes in this interval. Here we report mutations in one of these genes, Nsdhl, encoding an NAD(P)H steroid dehydrogenase-like protein, in two independent Bpa and three independent Str alleles. Quantitative analysis of sterols from tissues of affected Bpa mice support a role for Nsdhl in cholesterol biosynthesis. Our results demonstrate that Bpa and Str are allelic mutations and identify the first mammalian locus associated with an X-linked dominant, male-lethal phenotype. They also expand the spectrum of phenotypes associated with abnormalities of cholesterol metabolism.

Residues that influence in vivo and in vitro CbbR function in Rhodobacter sphaeroides and identification of a specific region critical for co‐inducer recognition

CbbR is a LysR‐type transcriptional regulator (LTTR) that is required to activate transcription of the cbb operons, responsible for CO2 fixation, in Rhodobacter sphaeroides. LTTR proteins often require a co‐inducer to regulate transcription. Previous studies suggested that ribulose 1,5‐bisphosphate (RuBP) is a positive effector for CbbR function in this organism. In the current study, RuBP was found to increase the electrophoretic mobility of the CbbR/cbbI promoter complex. To define and analyse the co‐inducer recognition region of CbbR, constitutively active mutant CbbR proteins were isolated. Under growth conditions that normally maintain transcriptionally inactive cbb operons, the mutant CbbR proteins activated transcription. Fourteen of the constitutively active mutants resulted from a single amino acid substitution. One mutant was derived from amino acid substitutions at two separate residues that appeared to act synergistically. Different mutant proteins showed both sensitivity and insensitivity to RuBP and residues that conferred constitutive transcriptional activity could be highlighted on a three‐dimensional model, with several residues unique to CbbR shown to be at locations critical to LTTR function. Many of the constitutive residues clustered in or near two specific loops in the LTTR tertiary structure, corresponding to a proposed site of co‐inducer binding.

Protein–protein interactions between CbbR and RegA (PrrA), transcriptional regulators of the cbb operons of Rhodobacter sphaeroides

CbbR and RegA (PrrA) are transcriptional regulators of the cbbI and cbbII (Calvin–Benson–Bassham CO2 fixation pathway) operons of Rhodobacter sphaeroides. Both proteins interact specifically with promoter sequences of the cbb operons. RegA has four DNA binding sites within the cbbI promoter region, with the CbbR binding site and RegA binding site 1 overlapping each other. This study demonstrated that CbbR and RegA interact and form a discrete complex in vitro, as illustrated by gel mobility shift experiments, direct isolation of the proteins from DNA complexes, and chemical cross‐linking analyses. For CbbR/RegA interactions to occur, CbbR must be bound to the DNA, with the ability of CbbR to bind the cbbI promoter enhanced by RegA. Conversely, interactions with CbbR did not require RegA to bind the cbbI promoter. RegA itself formed incrementally larger multimeric complexes with DNA as the concentration of RegA increased. The presence of RegA binding sites 1, 2 and 3 promoted RegA/DNA binding at significantly lower concentrations of RegA than when RegA binding site 3 was not present in the cbbI promoter. These studies support the premise that both CbbR and RegA are necessary for optimal transcription of the cbbI operon genes of R. sphaeroides.

Nuclear Interactions Are Necessary for Translational Enhancement by Spleen Necrosis Virus RU5

ABSTRACT The 5′ long terminal repeat of spleen necrosis virus (SNV) facilitates Rev/Rev-responsive element (RRE)-independent expression of intron-containing human immunodeficiency virus type 1 (HIV-1) gag. The SNV RU5 region, which corresponds to the 165-nucleotide 5′ RNA terminus, functions in a position- and orientation-dependent manner to enhance polysome association of intron-containing HIV-1 gag RNA and also nonviral luc RNA. Evidence is mounting that association with nuclear factors during intron removal licenses mRNAs for nuclear export, efficient translation, and nonsense-mediated decay. This project addressed the relationship between the nuclear export pathway of SNV RU5-reporter RNA and translational enhancement. Results of RNA transfection experiments suggest that cytoplasmic proteins are insufficient for SNV RU5 translational enhancement of gag or luc RNA. Reporter gene assays, leptomycin B (LMB) sensitivity experiments, and RNase protection assays indicate that RU5 gag RNA accesses a nuclear export pathway that is distinct from the LMB-inhibited leucine-rich nuclear export sequence-dependent CRM1 pathway, which is used by the HIV-1 RRE. As a unique tool with which to investigate the relationship between different RNA trafficking routes and translational enhancement, SNV RU5 and Rev/RRE were combined on a single gag RNA. We observed a less-than-synergistic effect on cytoplasmic mRNA utilization. Instead, Rev/RRE diverts RU5 gag RNA to the CRM1-dependent, LMB-inhibited pathway and abrogates translational enhancement by SNV RU5. Our study is the first to show that a nuclear factor(s) directs SNV RU5-containing RNAs to a distinct export pathway that is not inhibited by LMB and programs the intron-containing transcript for translational enhancement.

CbbR, the Master Regulator for Microbial Carbon Dioxide Fixation

Biological carbon dioxide fixation is an essential and crucial process catalyzed by both prokaryotic and eukaryotic organisms to allow ubiquitous atmospheric CO2 to be reduced to usable forms of organic carbon. This process, especially the Calvin-Bassham-Benson (CBB) pathway of CO2 fixation, provides the bulk of organic carbon found on earth. The enzyme ribulose 1,5-bisphosphate (RuBP) carboxylase/oxygenase (RubisCO) performs the key and rate-limiting step whereby CO2 is reduced and incorporated into a precursor organic metabolite. This is a highly regulated process in diverse organisms, with the expression of genes that comprise the CBB pathway (the cbb genes), including RubisCO, specifically controlled by the master transcriptional regulator protein CbbR. Many organisms have two or more cbb operons that either are regulated by a single CbbR or employ a specific CbbR for each cbb operon. CbbR family members are versatile and accommodate and bind many different effector metabolites that influence CbbRs ability to control cbb transcription. Moreover, two members of the CbbR family are further posttranslationally modified via interactions with other transcriptional regulator proteins from two-component regulatory systems, thus augmenting CbbR-dependent control and optimizing expression of specific cbb operons. In addition to interactions with small effector metabolites and other regulator proteins, CbbR proteins may be selected that are constitutively active and, in some instances, elevate the level of cbb expression relative to wild-type CbbR. Optimizing CbbR-dependent control is an important consideration for potentially using microbes to convert CO2 to useful bioproducts.

Activation of conventional kinesin motors in clusters by Shaw voltage-gated K+ channels

Summary The conventional kinesin motor transports many different cargos to specific locations in neurons. How cargos regulate motor function remains unclear. Here we focus on KIF5, the heavy chain of conventional kinesin, and report that the Kv3 (Shaw) voltage-gated K+ channel, the only known tetrameric KIF5-binding protein, clusters and activates KIF5 motors during axonal transport. Endogenous KIF5 often forms clusters along axons, suggesting a potential role of KIF5-binding proteins. Our biochemical assays reveal that the high-affinity multimeric binding between the Kv3.1 T1 domain and KIF5B requires three basic residues in the KIF5B tail. Kv3.1 T1 competes with the motor domain and microtubules, but not with kinesin light chain 1 (KLC1), for binding to the KIF5B tail. Live-cell imaging assays show that four KIF5-binding proteins, Kv3.1, KLC1 and two synaptic proteins SNAP25 and VAMP2, differ in how they regulate KIF5B distribution. Only Kv3.1 markedly increases the frequency and number of KIF5B-YFP anterograde puncta. Deletion of Kv3.1 channels reduces KIF5 clusters in mouse cerebellar neurons. Therefore, clustering and activation of KIF5 motors by Kv3 regulate the motor number in carrier vesicles containing the channel proteins, contributing not only to the specificity of Kv3 channel transport, but also to the cargo-mediated regulation of motor function.

Integrative Control of Carbon, Nitrogen, Hydrogen, and Sulfur Metabolism: The Central Role of the Calvin–Benson–Bassham Cycle

Nonsulfur purple (NSP) photosynthetic bacteria use the Calvin-Benson-Bassham (CBB) reductive pentose phosphate pathway for the reduction of CO(2) via ribulose 1,5-bisphosphate (RuBP) carboxylase-oxygenase (RubisCO), as a means to build cell mass during chemoautotrophic or photoautotrophic conditions. In addition, the CBB pathway plays an important role in maintaining redox balance during photoheterotrophic growth conditions. In this communication we describe protein-protein interactions between two transcriptional regulators CbbR and RegA and the possible role of the CbbX protein in regulating the CBB pathway in Rhodobacter sphaeroides. In Rhodopseudomonas palustris, the CbbR and the CbbRRS system (a three-protein, two-component regulatory system) regulate the CBB pathway. Moreover, derepression of the nitrogenase complex, and the production of hydrogen gas, appears to be a common mechanism to balance the redox potential in RubisCO-compromised strains of NSP photosynthetic bacteria.

Amino Acid Residues of RegA Important for Interactions with the CbbR-DNA Complex of Rhodobacter sphaeroides

CbbR and RegA (PrrA) are transcriptional regulators of the Calvin-Benson-Bassham (CBB) CO2 fixation pathway (cbbI and cbbII) operons of Rhodobacter sphaeroides. The CbbR and RegA proteins interact, but CbbR must be bound to the promoter DNA in order for RegA-CbbR protein-protein interactions to occur. RegA greatly enhances the ability of CbbR to bind the cbbI promoter or greatly enhances the stability of the CbbR/promoter complex. The N-terminal receiver domain and the DNA binding domain of RegA were shown to interact with CbbR. Residues in α-helix 7 and α-helix 8 of the DNA binding domain (helix-turn-helix) of RegA directly interacted with CbbR, with α-helix 7 positioned immediately above the DNA and α-helix 8 located in the major groove of the DNA. A CbbR protein containing only the DNA binding motif and the linker helix was capable of binding to RegA. In contrast, a truncated CbbR containing only the linker helix and recognition domains I and II (required for effector binding) was not able to interact with RegA. The accumulated results strongly suggest that the DNA binding domains of both proteins interact to facilitate optimal transcriptional control over the cbb operons. In vivo analysis, using constitutively active mutant CbbR proteins, further indicated that CbbR must interact with phosphorylated RegA in order to accomplish transcriptional activation.

Superinfecting Activity of the B95–8 Isolate of Epstein-Barr Virus

The B95–8 isolate of Epstein-Barr virus (EBV) has been characterized as a transforming strain (1). In contrast, the P3HR-1 (HR-1) isolate has been shown to superinfect latently infected cells such as Raji and NC-37 cells (2), Differences in the activities of these strains of EBV have been thought to be, at least in part, due to specific characteristic deletions in the viral genomes (3). A third isolate of EBV, derived from an epithelial (Ad-AH)/epithelial (NPC tumor) hybrid designated NPC-KT has been shown to have both transforming and lytic biological properties (4,5). Recent work in our laboratory has shown that, by using 100x concentrates of culture supernatants, superinfecting activity can be demonstrated for the B95–8 isolate of EBV.