bluuebunny/crossref_metadata_2025 huggingface dataset:
Neutral behavior of shared polymorphism
Authors: Clark Andrew G.
Published: July 2002

Several cases have been described in the literature where genetic
polymorphism appears to be shared between a pair of species. Here we
examine the distribution of times to random loss of shared polymorphism
in the context of the neutral Wright–Fisher model. Order statistics
are used to obtain the distribution of times to loss of a shared
polymorphism based on Kimura’s solution to the diffusion approximation
of the Wright–Fisher model. In a single species, the expected
absorption time for a neutral allele having an initial allele frequency
of ½ is 2.77
N
generations. If two species
initially share a polymorphism, that shared polymorphism is lost as
soon as either of two species undergoes fixation. The loss of a shared
polymorphism thus occurs sooner than loss of polymorphism in a single
species and has an expected time of 1.7
N
generations.
Molecular sequences of genes with shared polymorphism may be
characterized by the count of the number of sites that segregate in
both species for the same nucleotides (or amino acids). The
distribution of the expected numbers of these shared polymorphic sites
also is obtained. Shared polymorphism appears to be more likely at
genetic loci that have an unusually large number of segregating
alleles, and the neutral coalescent proves to be very useful in
determining the probability of shared allelic lineages expected by
chance. These results are related to examples of shared polymorphism in
the literature.
Significance of chaperonin 10-mediated inhibition of ATP hydrolysis by chaperonin 60
Authors: Dubaquié Yves , Looser Renate , Rospert Sabine
Published: July 2002

Chaperonins are essential for the folding of proteins in bacteria, mitochondria, and chloroplasts. We have functionally characterized the yeast mitochondrial chaperonins hsp60 and hsp10. In the presence of ADP, one molecule of hsp10 binds to hsp60 with an apparent
K
d
of 0.9 nM and a second molecule of hsp10 binds with a
K
d
of 24 nM. In the presence of ATP, the purified yeast chaperonins mediate the refolding of mitochondrial malate dehydrogenase. Hsp10 inhibits the ATPase activity of hsp60 by about 40%. Hsp10(P36H) is a point mutant of hsp10 that confers temperature-sensitive growth to yeast. Consistent with the
in vivo
phenotype, refolding of mitochondrial malate dehydrogenase in the presence of purified hsp10(P36H) and hsp60 is reduced at 25°C and abolished at 30°C. The affinity of hsp10(P36H) to hsp60 as well as to
Escherichia coli
GroEL is reduced. However, this decrease in affinity does not correlate with the functional defect, because hsp10(P36H) fully assists the GroEL-mediated refolding of malate dehydrogenase at 30°C. Refolding activity, rather, correlates with the ability of hsp10(P36H) to inhibit the ATPase of GroEL but not that of hsp60. Based on our findings, we propose that the inhibition of ATP hydrolysis is mechanistically coupled to chaperonin-mediated protein folding.
A substance P (neurokinin-1) receptor mutant carboxyl-terminally truncated to resemble a naturally occurring receptor isoform displays enhanced responsiveness and resistance to desensitization
Authors: Li Hanzhong , Leeman Susan E. , Slack Barbara E. , Hauser George , Saltsman Wayne S. , Krause James E. , Blusztajn Jan Krzysztof , Boyd Norman D.
Published: July 2002

Two isoforms of the substance P (SP) receptor, differing in the length of the cytoplasmic carboxyl-terminus by ≈8 kDa, have been detected previously in rat salivary glands and other tissues. The binding and functional properties of these two isoforms have been investigated using full-length (407 amino acids) and carboxyl-terminally truncated (324 amino acids) rat SP receptors transfected stably into Chinese hamster ovary cells. Both the full-length and the truncated receptor bound radiolabeled SP with a similar
K
d
(≈0.1 nM). The average number of high affinity SP binding sites per cell was 1.0 × 10
5
and 0.3 × 10
5
for the full-length and the truncated SP receptor, respectively. In both cell lines, SP induced a rapid but transient increase in cytosolic calcium concentration ([Ca
2+
]
i
), which consisted of the release of Ca
2+
from intracellular stores and the influx of extracellular Ca
2+
. Both components are dependent on phospholipase C activation. Although the full-length and the truncated receptor utilize the same calcium pathways, they differ in their EC
50
values (0.28 nM for the full-length; 0.07 nM for the truncated). These differences in responsiveness may be related to the observed differences in receptor desensitization. The truncated receptor, in contrast to the full-length receptor, does not undergo rapid and long-lasting desensitization. Cells possessing the short isoform of the SP receptor would thus be expected to exhibit a prolonged responsiveness.
In vitro
properties of the first ORF protein from mouse LINE-1 support its role in ribonucleoprotein particle formation during retrotransposition
Authors: Kolosha Vladimir O. , Martin Sandra L.
Published: July 2002

LINEs are transposable elements, widely distributed among eukaryotes, that move via reverse transcription of an RNA intermediate. Mammalian LINEs have two ORFs (ORF1 and ORF2). The proteins encoded by these ORFs play important roles in the retrotransposition process. Although the predicted amino acid sequence of ORF1 is not closely related to any known proteins, it is highly basic; thus, it has long been hypothesized that ORF1 protein functions to bind LINE-1 (L1) RNA during retrotransposition. Cofractionation of ORF1 protein and L1 RNA in extracts from both mouse and human embryonal carcinoma cells indicated that ORF1 protein binds L1 RNA, forming a ribonucleoprotein particle. Based on UV crosslinking and electrophoretic mobility-shift assays using purified components, we demonstrate here that the ORF1 protein encoded by mouse L1 binds nucleic acids with a strong preference for RNA and other single-stranded nucleic acids. Furthermore, multiple copies of ORF1 protein appear to bind single-stranded nucleic acid in a manner suggesting positive cooperativity; such binding characteristics are likely to be facilitated by the protein–protein interactions detected among molecules of ORF1 polypeptide by coimmunoprecipitation. These observations are consistent with the formation of ribonucleoprotein particles containing L1 RNA and ORF1 protein and provide additional evidence for the role of ORF1 protein during retrotransposition of L1.
Regulation of sulfur assimilation in higher plants: A sulfate transporter induced in sulfate-starved roots plays a central role in
Arabidopsis thaliana
Authors: Takahashi Hideki , Yamazaki Mami , Sasakura Noriko , Watanabe Akiko , Leustek Thomas , Engler Janice de Almeida , Engler Gilbert , Van Montagu Marc , Saito Kazuki
Published: July 2002

Proton/sulfate cotransporters in the plasma membranes are responsible for uptake of the environmental sulfate used in the sulfate assimilation pathway in plants. Here we report the cloning and characterization of an
Arabidopsis thaliana
gene,
AST68
, a new member of the sulfate transporter gene family in higher plants. Sequence analysis of cDNA and genomic clones of
AST68
revealed that the
AST68
gene is composed of 10 exons encoding a 677-aa polypeptide (74.1 kDa) that is able to functionally complement a
Saccharomyces cerevisiae
mutant lacking a sulfate transporter gene. Southern hybridization and restriction fragment length polymorphism mapping confirmed that
AST68
is a single-copy gene that maps to the top arm of chromosome 5. Northern hybridization analysis of sulfate-starved plants indicated that the steady-state mRNA abundance of
AST68
increased specifically in roots up to 9-fold by sulfate starvation.
In situ
hybridization experiments revealed that
AST68
transcripts were accumulated in the central cylinder of sulfate-starved roots, but not in the xylem, endodermis, cortex, and epidermis. Among all the structural genes for sulfate assimilation, sulfate transporter (
AST68
), APS reductase (
APR1
), and serine acetyltransferase (
SAT1
) were inducible by sulfate starvation in
A. thaliana
. The sulfate transporter (
AST68
) exhibited the most intensive and specific response in roots, indicating that
AST68
plays a central role in the regulation of sulfate assimilation in plants.
Functional analysis of DNA bending and unwinding by the high mobility group domain of LEF-1
Authors: Giese Klaus , Pagel John , Grosschedl Rudolf
Published: July 2002

LEF-1 (lymphoid enhancer-binding factor 1) is a cell type-specific member of the family of high mobility group (HMG) domain proteins that recognizes a specific nucleotide sequence in the T cell receptor (TCR) α enhancer. In this study, we extend the analysis of the DNA-binding properties of LEF-1 and examine their contributions to the regulation of gene expression. We find that LEF-1, like nonspecific HMG-domain proteins, can interact with irregular DNA structures such as four-way junctions, albeit with lower efficiency than with specific duplex DNA. We also show by a phasing analysis that the LEF-induced DNA bend is directed toward the major groove. In addition, we find that the interaction of LEF-1 with a specific binding site in circular DNA changes the linking number of DNA and unwinds the double helix. Finally, we identified two nucleotides in the LEF-1-binding site that are important for protein-induced DNA bending. Mutations of these nucleotides decrease both the extent of DNA bending and the transactivation of the TCRα enhancer by LEF-1, suggesting a contribution of protein-induced DNA bending to the function of TCRα enhancer.
Transcriptional regulation in Archaea:
In vivo
demonstration of a repressor binding site in a methanogen
Authors: Cohen-Kupiec Rachel , Blank Carrine , Leigh John A.
Published: July 2002

The status of the Archaea as one of the three primary Domains emphasizes the importance of understanding their molecular fundamentals. Basic transcription in the Archaea resembles eucaryal transcription. However, little is known about transcriptional regulation. We have taken an
in vivo
approach, using genetics to address transcriptional regulation in the methanogenic Archaeon
Methanococcus maripaludis
. We identified a repressor binding site that regulates
nif
(nitrogen fixation) gene expression. The repressor binding site was palindromic (an inverted repeat) and was located just after the transcription start site of
nifH
. Mutations that changed the sequence of the palindrome resulted in marked decreases in repression by ammonia, even when the palindromic nature of the site was retained. The same mutations greatly decreased binding to the site by components of cell extract. These results provide the first partial description of a transcriptional regulatory mechanism in the methanogenic Archaea. This work also illustrates the utility of genetic approaches in
Methanococcus
that have not been widely used in the methanogens: directed mutagenesis and reporter gene fusions with
lacZ
.
Zinc porphyrins: Potent inhibitors of hematopoieses in animal and human bone marrow
Authors: Lutton John D. , Abraham Nader G. , Drummond George S. , Levere Richard D. , Kappas Attallah
Published: July 2002

The effects of selected heme analogues on heme oxygenase activity in tissues and on human and rabbit bone marrow hematopoietic colony growth were examined. Zinc protoporphyrin (ZnPP) and zinc mesoporphyrin (ZnMP), at concentrations ranging between 1 and 20 μM, produced significant inhibition of human and rabbit bone marrow erythroid (CFU-E, BFU-E) and myeloid (CFU-GM) colony growth. The growth inhibition produced by ZnPP or ZnMP was not overcome with exposure of cultures to elevated levels of the growth factors erythropoietin and granulocyte–macrophage colony stimulating factor. In contrast, tin protoporphyrin and tin mesoporphyrin did not display any significant bone marrow toxicity when used at similar concentrations. In other studies, differential effects of tin mesoporphyrin and ZnMP administered intravenously on kidney heme oxygenase were demonstrated. Chromium mesoporphyrin administered intravenously proved lethal to animals. These results indicate that exposure of bone marrow to ZnPP and ZnMP can be deleterious to hematopoietic cells and raise the possibility that ZnPP, which is endogenously formed and found in high concentration in red blood cells in lead-poisoned children, may itself participate in the bone marrow toxicity produced by this metal.
DNA damage can alter the stability of nucleosomes: Effects are dependent on damage type
Authors: Mann David B. , Springer David L. , Smerdon Michael J.
Published: July 2002

We have investigated the effects of DNA damage by (±)-
anti
-benzo[
a
]pyrene diol epoxide (BPDE) and UV light on the formation of a positioned nucleosome in the
Xenopus borealis
5S rRNA gene. Gel-shift analysis of the reconstituted products indicates that BPDE damage facilitates the formation of a nucleosome onto this sequence. Competitive reconstitution experiments show that average levels of 0.5, 0.9, and 2.1 BPDE adducts/146 bp of 5S DNA (i.e., the size of DNA associated with a nucleosome core particle) yield changes of −220, −290, and −540 cal/mol, respectively, in the free energy (ΔG) of nucleosome formation. These values yield increases of core histone binding to 5S DNA (K
a
) of 1.4-, 1.6-, and 2.5-fold, compared with undamaged DNA. Conversely, irradiation with UV light decreases nucleosome formation. Irradiation at either 500 or 2500 J/m
2
of UV light [0.6 and 0.8 cyclobutane pyrimidine dimer/146 bp (on average), respectively] results in respective changes of +130 and +250 cal/mol. This translates to decreases in core histone binding to irradiated 5S DNA (K
a
) of 1.2- and 1.5-fold compared with undamaged DNA. These results indicate that nucleosome stability can be markedly affected by the formation of certain DNA lesions. Such changes could have major effects on the kinetics of DNA processing events.
Transcriptional analysis of nucleolar dominance in polyploid plants: Biased expression/silencing of progenitor rRNA genes is developmentally regulated in
Brassica
Authors: Chen Z. Jeffrey , Pikaard Craig S.
Published: July 2002

Nucleolar dominance is an epigenetic phenomenon that describes the formation of nucleoli around rRNA genes inherited from only one parent in the progeny of an interspecific hybrid. Despite numerous cytogenetic studies, little is known about nucleolar dominance at the level of rRNA gene expression in plants. We used S1 nuclease protection and primer extension assays to define nucleolar dominance at a molecular level in the plant genus
Brassica
. rRNA transcription start sites were mapped in three diploids and in three allotetraploids (amphidiploids) and one allohexaploid species derived from these diploid progenitors. rRNA transcripts of only one progenitor were detected in vegetative tissues of each polyploid. Dominance was independent of maternal effect, ploidy, or rRNA gene dosage. Natural and newly synthesized amphidiploids yielded the same results, arguing against substantial evolutionary effects. The hypothesis that nucleolar dominance in plants is correlated with physical characteristics of rRNA gene intergenic spacers is not supported in
Brassica
. Furthermore, in
Brassica napus
, rRNA genes silenced in vegetative tissues were found to be expressed in all floral organs, including sepals and petals, arguing against the hypothesis that passage through meiosis is needed to reactivate suppressed genes. Instead, the transition of inflorescence to floral meristem appears to be a developmental stage when silenced genes can be derepressed.
The conformation of NAD
+
bound to lactate dehydrogenase determined by nuclear magnetic resonance with suppression of spin diffusion
Authors: Vincent Sébastien J. F. , Zwahlen Catherine , Post Carol Beth , Burgner John W. , Bodenhausen Geoffrey
Published: July 2002

We have reinvestigated the conformation of NAD
+
bound to dogfish lactate dehydrogenase (LDH) by using an NMR experiment that allows one to exploit nuclear Overhauser effects to determine internuclear distances between pairs of protons, without perturbation of spin-diffusion effects from other protons belonging either to the cofactor or to the binding pocket of the enzyme. The analysis indicates that the structure of bound NAD
+
is in accord with the conformation determined in the solid state by x-ray diffraction for the adenosine moiety, but deviates significantly from that of the nicotinamide. The NMR data indicate conformational averaging about the glycosidic bond of the nicotinamide nucleotide. In view of the strict stereospecificity of catalysis by LDH and the conformational averaging of bound NAD
+
that we infer from solution-state NMR, we suggest that LDH binds the cofactor in both
syn
and
anti
conformations, but that binding interactions in the
syn
conformation are not catalytically productive.
Kinetics of force generation by single kinesin molecules activated by laser photolysis of caged ATP
Authors: Higuchi Hideo , Muto Etsuko , Inoue Yuichi , Yanagida Toshio
Published: July 2002

To relate transients of force by single kinesin molecules with the elementary steps of the ATPase cycle, we measured the time to force generation by kinesin after photorelease of ATP from caged ATP. Kinesin-coated beads were trapped by an infrared laser and brought onto microtubules fixed to a coverslip. Tension was applied to a kinesin-microtubule rigor complex using the optical trap, and ATP was released by flash photolysis of caged ATP with a UV laser. Kinesin started to generate force and move stepwise with a step size of 8 nm at average times of 31, 45, and 79 ms after photorelease of 450, 90, and 18 μM ATP, respectively. The kinetics of force generation were consistent with a two-step reaction: ATP binding, with an apparent second-order rate constant of 0.7 μM
−1
·s
−1
, followed by force generation at 45 s
−1
per kinesin molecule. The transient rate of force generation was close to the rate of the ATPase cycle in solution, suggesting that the rate-limiting step of ATPase cycle is involved with the force generation.
Functional and idling rotatory motion within F
1
-ATPase
Authors: Sabbert D. , Engelbrecht S. , Junge W.
Published: July 2002

ATP synthase mediates proton flow through its membrane portion, F
0
, which drives the synthesis of ATP in its headpiece, F
1
. The F
1
-portion contains a hexagonal array of three subunits α and three β encircling a central subunit γ, that in turn interacts with a smaller ɛ and with F
0
. Recently we reported that the application of polarized absorption recovery after photobleaching showed the ATP-driven rotation of γ over at least two, if not three, β. Here we extend probes of such rotation aided by a new theory for assessing continuous versus stepped, Brownian versus unidirectional molecular motion. The observed relaxation of the absorption anisotropy is fully compatible with a unidirectional and stepping rotation of γ over three equidistantly spaced angular positions in the hexagon formed by the alternating subunits α and β. The results strongly support a rotational catalysis with equal participation of all three catalytic sites. In addition we report a limited rotation of γ without added nucleotides, perhaps idling and of Brownian nature, that covers only a narrow angular domain.
Persistence of an alternate chromatin structure at silenced loci in the absence of silencers
Authors: Cheng Tzu-Hao , Li Yao-Cheng , Gartenberg Marc R.
Published: July 2002

In
Saccharomyces cerevisiae
, genes placed near telomeres or the silent
HML
and
HMR
mating-type loci are transcriptionally repressed by a heterochromatin-like structure. We have generated nonreplicating DNA rings by recombination
in vivo
to examine the role of chromosomal context on transcriptional repression. Specifically, recombination at
HMR
was used to produce rings that lacked the E and I silencers. An altered level of DNA supercoiling was observed in these rings but not in comparable rings from derepressed loci. Our results indicate that a repressive chromatin structure persists in an extrachromosomal environment immediately following removal of the
cis
-acting control elements. Examination of both chromatin footprints and DNA sequence dependence revealed that changes in nucleosome number could account for the topology shifts. Upon continued cell growth, the differences in supercoiling were lost and transcriptional competence was restored. These results show that silencers are required for sustained persistence of repressive chromatin structure, even in the absence of DNA replication.
Identification of a cAMP-dependent protein kinase catalytic subunit required for virulence and morphogenesis in
Ustilago maydis
Authors: Dürrenberger Franz , Wong Katherine , Kronstad James W.
Published: July 2002

Ustilago maydis
, a fungal pathogen of maize, alternates between budding and filamentous growth in response to mating and other environmental signals. Defects in components of the cAMP signaling pathway affect this morphological transition and reveal an association of budding growth with elevated cAMP levels and filamentous growth with low cAMP levels. We have identified two genes,
adr1
and
uka1
, encoding catalytic subunits of cAMP-dependent protein kinase (PKA). Disruption of
adr1
resulted in a constitutively filamentous growth phenotype similar to that of mutants deficient in adenylyl cyclase. Importantly,
adr1
is required for pathogenicity and is responsible for the majority of PKA activity in fungal cells. In contrast,
uka1
has little influence on pathogenicity, and deletion of the
uka1
gene does not affect cell morphology. These results provide compelling evidence that regulated PKA activity is crucial during infectious development of
U. maydis
.
Characterization of MADS homeotic genes in the fern
Ceratopteris richardii
Authors: Hasebe Mitsuyasu , Wen Chi-Kuang , Kato Masahiro , Banks Jo Ann
Published: July 2002

The MADS genes encode a family of transcription factors, some of which control the identities of floral organs in flowering plants. To understand the role of MADS genes in the evolution of floral organs, five MADS genes (CMADS1, 2, 3, 4, and 6) were cloned from the fern
Ceratopteris richardii
, a nonflowering plant. A gene tree of partial amino acid sequences of seed plant and fern MADS genes showed that the fern genes form three subfamilies. All members of one of the fern MADS subfamilies have additional amino-terminal amino acids, which is a synapomorphic character of the
AGAMOUS
subfamily of the flowering plant MADS genes. Their structural similarity indicates a sister relationship between the two subfamilies. The temporal and spatial patterns of expression of the five fern MADS genes were assessed by Northern blot analyses and
in situ
hybridizations. CMADS1, 2, 3, and 4 are expressed similarly in the meristematic regions and primordia of sporophyte shoots and roots, as well as in reproductive structures, including sporophylls and sporangial initials, although the amount of expression in each tissue is different in each gene. CMADS6 is expressed in gametophytic tissues but not in sporophytic tissues. The lack of organ-specific expression of MADS genes in the reproductive structures of the fern sporophyte may indicate that the restriction of MADS gene expression to specific reproductive organs and the specialization of MADS gene functions as homeotic selector genes in the flowering plant lineage were important in floral organ evolution.
Systematic identification of essential genes by
in vitro mariner
mutagenesis
Authors: Akerley Brian J. , Rubin Eric J. , Camilli Andrew , Lampe David J. , Robertson Hugh M. , Mekalanos John J.
Published: July 2002

Although the complete DNA sequences of several microbial genomes are now available, nearly 40% of the putative genes lack identifiable functions. Comprehensive screens and selections for identifying functional classes of genes are needed to convert sequence data into meaningful biological information. One particularly significant group of bacterial genes consists of those that are essential for growth or viability. Here, we describe a simple system for performing transposon mutagenesis on naturally transformable organisms along with a technique to rapidly identify essential or conditionally essential DNA segments. We show the general utility of this approach by applying it to two human pathogens,
Haemophilus influenzae
and
Streptococcus pneumoniae
, in which we detected known essential genes and assigned essentiality to several ORFs of unknown function.
Genetically engineered broad-spectrum disease resistance in tomato
Authors: Oldroyd Giles E. D. , Staskawicz Brian J.
Published: July 2002

Resistance in tomato to the bacterial pathogen
Pseudomonas syringae pathovar tomato
requires
Pto
and
Prf
. Mutations that eliminate
Prf
show a loss of both
Pto
resistance and sensitivity to the organophosphate insecticide fenthion, suggesting that
Prf
controls both phenotypes. Herein, we report that the overexpression of
Prf
leads to enhanced resistance to a number of normally virulent bacterial and viral pathogens and leads to increased sensitivity to fenthion. These plants express levels of salicylic acid comparable to plants induced for systemic acquired resistance (SAR) and constitutively express pathogenesis related genes. These results suggest that the overexpression of
Prf
activates the
Pto
and
Fen
pathways in a pathogen-independent manner and leads to the activation of SAR. Transgene-induced SAR has implications for the generation of broad spectrum disease resistance in agricultural crop plants.
HIV, but not murine leukemia virus, vectors mediate high efficiency gene transfer into freshly isolated G
0
/G
1
human hematopoietic stem cells
Authors: Uchida Nobuko , Sutton Richard E. , Friera Annabelle M. , He Dongping , Reitsma Michael J. , Chang Wei Chun , Veres Gabor , Scollay Roland , Weissman Irving L.
Published: July 2002

Recent studies have opened the possibility that quiescent, G
0
/G
1
hematopoietic stem cells (HSC) can be gene transduced; lentiviruses (such as HIV type 1, HIV) encode proteins that permit transport of the viral genome into the nucleus of nondividing cells. We and others have recently demonstrated efficient transduction by using an HIV-1-based vector gene delivery system into various human cell types including human CD34
+
cells or terminally differentiated neurons. Here we compare the transduction efficiency of two vectors, HIV-based and murine leukemia virus (MuLV)-based vectors, on untreated and highly purified human HSC subsets that are virtually all in G
0
/G
1
. The HIV vector, but not MuLV vector supernatants, transduced freshly isolated G
0
/G
1
HSC from mobilized peripheral blood. Single-step transduction using replication-defective HIV resulted in HSC that expressed the green fluorescent protein (GFP) transgene while retaining their stem cell phenotype; clonal outgrowths of these GFP
+
HSC on bone marrow stromal cells fully retained GFP expression for at least 5 weeks. MuLV-based vectors did not transduce resting HSC, as measured by transgene expression, but did so readily when the HSC were actively cycling after culture
in vitro
for 3 days in a cytokine cocktail. These results suggest that resting HSC may be transduced by lentiviral-based, but not MuLV, vectors and maintain their primitive phenotype, pluripotentiality, and at least
in vitro
, transgene expression.
Inhibition of protease-resistant prion protein formation by porphyrins and phthalocyanines
Authors: Caughey Winslow S. , Raymond Lynne D. , Horiuchi Motohiro , Caughey Byron
Published: July 2002

A central aspect of pathogenesis in the transmissible spongiform encephalopathies or prion diseases is the conversion of normal protease-sensitive prion protein (PrP-sen) to the abnormal protease-resistant form, PrP-res. Here we identify porphyrins and phthalocyanines as inhibitors of PrP-res accumulation. The most potent of these tetrapyrroles had IC
50
values of 0.5–1 μM in scrapie-infected mouse neuroblastoma (ScNB) cell cultures. Inhibition was observed without effects on protein biosynthesis in general or PrP-sen biosynthesis in particular. Tetrapyrroles also inhibited PrP-res formation in a cell-free reaction composed predominantly of hamster PrP-res and PrP-sen. Inhibitors were found among phthalocyanines, deuteroporphyrins IX, and meso-substituted porphines; examples included compounds containing anionic, neutral protic, and cationic peripheral substituents and various metals. We conclude that certain tetrapyrroles specifically inhibit the conversion of PrP-sen to PrP-res without apparent cytotoxic effects. The inhibition observed in the cell-free conversion reaction suggests that the mechanism involved direct interactions of the tetrapyrrole with PrP-res and/or PrP-sen. These findings introduce a new class of inhibitors of PrP-res formation that represents a potential source of therapeutic agents for transmissible spongiform encephalopathies.
Galanin regulates prolactin release and lactotroph proliferation
Authors: Wynick David , Small Caroline J. , Bacon Andrea , Holmes Fiona E. , Norman Michael , Ormandy Christopher J. , Kilic Eser , Kerr Niall C. H. , Ghatei Mohammad , Talamantes Frank , Bloom Stephen R. , Pachnis Vassilis
Published: July 2002

The neuropeptide galanin is predominantly expressed by the lactotrophs (the prolactin secreting cell type) in the rodent anterior pituitary and in the median eminence and paraventricular nucleus of the hypothalamus. Prolactin and galanin colocalize in the same secretory granule, the expression of both proteins is extremely sensitive to the estrogen status of the animal. The administration of estradiol-17β induces pituitary hyperplasia followed by adenoma formation and causes a 3,000-fold increase in the galanin mRNA content of the lactotroph. To further study the role of galanin in prolactin release and lactotroph growth we now report the generation of mice carrying a loss-of-function mutation of the endogenous galanin gene. There is no evidence of embryonic lethality and the mutant mice grow normally. The specific endocrine abnormalities identified to date, relate to the expression of prolactin. Pituitary prolactin message levels and protein content of adult female mutant mice are reduced by 30–40% compared with wild-type controls. Mutant females fail to lactate and pups die of starvation/dehydration unless fostered onto wild-type mothers. Prolactin secretion in mutant females is markedly reduced at 7 days postpartum compared with wild-type controls with an associated failure in mammary gland maturation. There is an almost complete abrogation of the proliferative response of the lactotroph to high doses of estrogen, with a failure to up-regulate prolactin release, STAT5 expression or to increase pituitary cell number. These data further support the hypothesis that galanin acts as a paracrine regulator of prolactin expression and as a growth factor to the lactotroph.
Detection of targeted
GFP
-
Hox
gene fusions during mouse embryogenesis
Authors: Godwin Alan R. , Stadler H. Scott , Nakamura Kotoka , Capecchi Mario R.
Published: July 2002

The ability to use a vital cell marker to study mouse embryogenesis will open new avenues of experimental research. Recently, the use of transgenic mice, containing multiple copies of the jellyfish gene encoding the green fluorescent protein (GFP), has begun to realize this potential. Here, we show that the fluorescent signals produced by single-copy, targeted
GFP
in-frame fusions with two different murine
Hox
genes,
Hoxa1
and
Hoxc13
, are readily detectable by using confocal microscopy. Since
Hoxa1
is expressed early and
Hoxc13
is expressed late in mouse embryogenesis, this study shows that single-copy
GFP
gene fusions can be used through most of mouse embryogenesis. Previously, targeted
lac
Z gene fusions have been very useful for analyzing mouse mutants. Use of
GFP
gene fusions extends the benefits of targeted
lac
Z gene fusions by providing the additional utility of a vital marker. Our analysis of the
Hoxc13
GFPneo
embryos reveals GFP expression in each of the sites expected from analysis of
Hoxc13
lacZneo
embryos. Similarly,
Hoxa1
GFPneo
expression was detected in all of the sites predicted from RNA
in situ
analysis. GFP expression in the foregut pocket of
Hoxa1
GFPneo
embryos suggests a role for
Hoxa1
in foregut-mediated differentiation of the cardiogenic mesoderm.
Chromosomal arm replacement generates a high level of intraspecific polymorphism in the terminal inverted repeats of the linear chromosomal DNA of
Streptomyces ambofaciens
Authors: Fischer Gilles , Wenner Thomas , Decaris Bernard , Leblond Pierre
Published: July 2002

The chromosomal DNA of the bacteria
Streptomyces ambofaciens
DSM40697 is an 8-Mb linear molecule that ends in terminal inverted repeats (TIRs) of 210 kb. The sequences of the TIRs are highly variable between the different linear replicons of
Streptomyces
(plasmids or chromosomes). Two spontaneous mutant strains harboring TIRs of 480 and 850 kb were isolated. The TIR polymorphism seen is a result of the deletion of one chromosomal end and its replacement by 480 or 850 kb of sequence identical to the end of the undeleted chromosomal arm. Analysis of the wild-type sequences involved in these rearrangements revealed that a recombination event took place between the two copies of a duplicated DNA sequence. Each copy was mapped to one chromosomal arm, outside of the TIR, and encoded a putative alternative sigma factor. The two ORFs, designated
hasR
and
hasL
, were found to be 99% similar at the nucleotide level. The sequence of the chimeric regions generated by the recombination showed that the chromosomal structure of the mutant strains resulted from homologous recombination events between the two copies. We suggest that this mechanism of chromosomal arm replacement contributes to the rapid evolutionary diversification of the sequences of the TIR in
Streptomyces
.
Temperature, template topology, and factor requirements of archaeal transcription
Authors: Bell Stephen D. , Jaxel Christine , Nadal Marc , Kosa Peter F. , Jackson Stephen P.
Published: July 2002

Although Archaea are prokaryotic and resemble Bacteria morphologically, their transcription apparatus is remarkably similar to those of eukaryotic cell nuclei. Because some Archaea exist in environments with temperatures of around 100°C, they are likely to have evolved unique strategies for transcriptional control. Here, we investigate the effects of temperature and DNA template topology in a thermophilic archaeal transcription system. Significantly, and in marked contrast with characterized eucaryal systems, archaeal DNA template topology has negligible effect on transcription levels at physiological temperatures using highly purified polymerase and recombinant transcription factors. Furthermore, archaeal transcription does not require hydrolysis of the β-γ phosphoanhydride bond of ATP. However, at lower temperatures, negatively supercoiled templates are transcribed more highly than those that are positively supercoiled. Notably, the block to transcription on positively supercoiled templates at lowered temperatures is at the level of polymerase binding and promoter opening. These data imply that Archaea do not possess a functional homologue of transcription factor TFIIH, and that for the promoters studied, transcription is mediated by TATA box-binding protein, transcription factor TFB, and RNA polymerase alone. Furthermore, they suggest that the reduction of plasmid linking number by hyperthermophilic Archaea
in vivo
in response to cold shock is a mechanism to maintain gene expression under these adverse circumstances.
Kinetics of CD4+ T cell repopulation of lymphoid tissues after treatment of HIV-1 infection
Authors: Zhang Zhi-Qiang , Notermans Daan W. , Sedgewick Gerald , Cavert Winston , Wietgrefe Stephen , Zupancic Mary , Gebhard Kristin , Henry Keith , Boies Lawrence , Chen Zongming , Jenkins Marc , Mills Roger , McDade Hugh , Goodwin Carolyn , Schuwirth Caspar M. , Danner Sven A. , Haase Ashley T.
Published: July 2002

Potent combinations of antiretroviral drugs diminish the turnover of CD4+ T lymphocytes productively infected with HIV-1 and reduce the large pool of virions deposited in lymphoid tissue (LT). To determine to what extent suppression of viral replication and reduction in viral antigens in LT might lead correspondingly to repopulation of the immune system, we characterized CD4+ T lymphocyte populations in LT in which we previously had quantitated viral load and turnover of infected cells before and after treatment. We directly measured by quantitative image analysis changes in total CD4+ T cell counts, the CD45RA+ subset, and fractions of proliferating or apoptotic CD4+ T cells. Compared with normal controls, we documented decreased numbers of CD4+ T cells and increased proliferation and apoptosis. After treatment, proliferation returned to normal levels, and total CD4+ T and CD45RA+ cells increased. We discuss the effects of HIV-1 on this subset based on the concept that renewal mechanisms in the adult are operating at full capacity before infection and cannot meet the additional demand imposed by the loss of productively infected cells. The slow increases in the CD45RA+ CD4+ T cells are consistent with the optimistic conclusions that (i) renewal mechanisms have not been damaged irreparably even at relatively advanced stages of infection and (ii) CD4+ T cell populations can be partially restored by control of active replication without eradication of HIV-1.
Oxa1p, an essential component of the N-tail protein export machinery in mitochondria
Authors: Hell Kai , Herrmann Johannes M. , Pratje Elke , Neupert Walter , Stuart Rosemary A.
Published: July 2002

A number of nuclear encoded inner membrane proteins of mitochondria span the membrane in such a manner that their N termini are located in the intermembrane space. Many of these proteins attain this membrane orientation by undergoing an export step from the matrix across the inner membrane. This export process, which resembles bacterial N-tail export from energetic and topogenic signal requirements, is facilitated by Oxa1p, a protein that has homologues throughout prokaryotes and eukaryotes. Oxa1p, as we have previously shown, is required to export the N and C termini of the mitochondrially encoded pCoxII to the intermembrane space. We demonstrate here that imported nuclear encoded proteins physically interact with Oxa1p and depend on Oxa1p for efficient export of their N termini to the intermembrane space. Furthermore, Oxa1p interacts with nascent polypeptide chains synthesized in mitochondria, including the fully synthesized pCoxII and CoxIII species. Thus, Oxa1p represents a component of a general export machinery of the mitochondrial inner membrane.
Herpes simplex virus 1 induces and blocks apoptosis at multiple steps during infection and protects cells from exogenous inducers in a cell-type-dependent manner
Authors: Galvan Veronica , Roizman Bernard
Published: July 2002

Several publications have attested to the ability of herpes simplex viruses to protect cells against apoptosis. We investigated the ability of the virus to protect cells in continuous cultivation from apoptosis induced by the virus itself, and by other known inducers such as exposure to the tumor necrosis factor α (TNFα), antibody to Fas, C2-ceramide, osmotic shock (sorbitol), and thermal shock. The salient features of the results were that the virus was able to protect cells against apoptosis by all of the agents tested, and that apoptosis induced by the virus was a very early event that did not requirede novoexpression of viral genes. However, these events were cell-type specific. Thus: (i) The cell lines tested exhibited fragmented chromosomal DNA following infection with a virus lacking functional α4 and US3 genes encoding the major regulatory protein and a viral protein kinase, respectively, but not by wild-type virus. (ii) Wild-type virus protected subconfluent SK-N-SH but not HeLa cells against induction of apoptosis by anti-Fas antibody, TNFα, C2-ceramide, and thermal shock. Confluent SK-N-SH cells were not protected from osmotic shock-induced apoptosis by wild-type infection. (iii) Wild-type virus protected SK-N-SH but not HeLa cells against induction of apoptosis by sorbitol, anti-Fas antibody, or TNFα and C2-ceramide. (iv) Mutant HSV-1(HFEM)tsB7 at the nonpermissive temperature infects cells but the DNA is not released from capsids, and therefore viral gene expression is restricted to the function of viral proteins introduced into the cell along with the capsid containing the viral DNA. HSV-1(HFEM)tsB7 induced apoptosis in Vero cells but not in SK-N-SH cells infected and maintained at 39.5°C. (v) Tests of two caspase inhibitors showed that they blocked apoptosis induced by C2-ceramide and sorbitol, but were not able to block apoptosis induced by the virus lacking functional α4 and US3 genes. We conclude that HSV-1 triggers apoptosis at multiple metabolic checkpoints and in turn has evolved mechanisms to block apoptosis at each point and that some of the pathways of induction are shared with exogenous inducers tested in this study whereas others are not.
Differential patterns of acquired virulence genes distinguish
Salmonella
strains
Authors: Conner Christopher P. , Heithoff Douglas M. , Julio Steven M. , Sinsheimer Robert L. , Mahan Michael J.
Published: July 2002

Analysis of several
Salmonella typhimurium in vivo
-induced genes located in regions of atypical base composition has uncovered acquired genetic elements that cumulatively engender pathogenicity. Many of these regions are associated with mobile elements, encode predicted adhesin and invasin-like functions, and are required for full virulence. Some of these regions distinguish broad host range from host-adapted
Salmonella
serovars and may contribute to inherent differences in host specificity, tissue tropism, and disease manifestation. Maintenance of this archipelago of acquired sequence by selection in specific hosts reveals a fossil record of the evolution of pathogenic species.
Evidence that HetR protein is an unusual serine-type protease
Authors: Zhou Ruanbao , Wei Xingcheng , Jiang Nan , Li Huiguang , Dong Yuqing , Hsi Kuo-L. , Zhao Jindong
Published: July 2002

The
hetR
gene plays a very important role in cell differentiation of heterocystous cyanobacteria. To understand the mechanism of the
hetR
gene product in regulation of heterocyst differentiation, the recombinant HetR protein (rHetR) was overproduced in
Escherichia coli
. Purified rHetR was unstable and degraded easily in solution. Phenylmethanesulfonyl fluoride, a serine-type protease inhibitor, prevented the degradation and was shown to modify covalently rHetR. Dansyl fluoride (DnsF), another serine-type protease inhibitor, also covalently modifies rHetR as shown by electrophoresis and electroblotting of the labeled rHetR and by MS. The labeling of rHetR with phenylmethanesulfonyl fluoride and DnsF was at the same site of rHetR and required Ca
2+
. S179N-rHetR, a mutant protein from strain 216 of
Anabaena
PCC 7120, which cannot differentiate heterocysts because of the mutation, was also overproduced and characterized. Although S170N-rHetR still can be labeled with DnsF, no proteolysis was observed, suggesting that Ser179 is involved in proteolytic activity. DnsF-labeled rHetR was digested with trypsin, and the labeled peptide was isolated and sequenced. The labeled peptide matches a sequence from HetR. These results show that HetR is a protease.
Endothelial Ca
2+
waves preferentially originate at specific loci in caveolin-rich cell edges
Authors: Isshiki Masashi , Ando Joji , Korenaga Risa , Kogo Hiroshi , Fujimoto Toyoshi , Fujita Toshiro , Kamiya Akira
Published: July 2002

Stimulation of endothelial cells (ECs) with ATP evoked an increase in intracellular Ca
2+
concentration ([Ca
2+
]
i
). In a single bovine aortic EC, the [Ca
2+
]
i
rise started at a specific peripheral locus and propagated throughout the entire cell as a Ca
2+
wave. The initiation locus was constant upon repeated stimulation with ATP or other agonists (bradykinin and thrombin). The Ca
2+
wave was unaffected by the removal of extracellular Ca
2+
, demonstrating its dependence on intracellular Ca
2+
release. Microinjection of heparin into the cell inhibited the ATP-induced Ca
2+
responses, indicating that the Ca
2+
wave is at least partly mediated by the inositol 1,4,5-trisphosphate receptor. Immunofluorescence staining revealed that caveolin, a marker protein for caveolae, is distributed heterogeneously in the cell and that Ca
2+
waves preferentially originate at caveolin-rich cell edges. In contrast to caveolin, internalized transferrin and subunits of the clathrin-associated adaptor complexes such as adaptor protein-1 and -2 were diffusely distributed. Disruption of microtubules by Colcemid led to redistribution of caveolin away from the edges into the perinuclear center of the cell, and the ATP-induced [Ca
2+
]
i
increase was initiated on the rim of the centralized caveolin. Thus, caveolae may be involved in the initiation of ATP-induced Ca
2+
waves in ECs.
Homotypic interaction and multimerization of nucleocapsid protein of tomato spotted wilt tospovirus: Identification and characterization of two interacting domains
Authors: Uhrig J. F. , Soellick T.-R. , Minke C. J. , Philipp C. , Kellmann J.-W. , Schreier P. H.
Published: July 2002

The nucleocapsid protein (N) of tomato spotted wilt tospovirus (TSWV) plays a central role in the viral life cycle. With the aid of the yeast two-hybrid system and surface plasmon resonance analysis, homotypic interaction and multimerization of the N protein was detected. Analysis of deletion mutants identified two binding regions in the protein, located at the N terminus (amino acids 1–39) and the C terminus (amino acids 233–248), respectively, implying a “head-to-tail” interaction of the N terminus with the C terminus to form a multimeric chain. Further characterization of the binding domains was performed by site-directed mutagenesis. Two phenylalanines (F242 and F246) highly conserved in the N proteins within the
Tospovirus
genus were shown to play a crucial role in the interaction.
Solution structure and dynamics of a
de novo
designed three-helix bundle protein
Authors: Walsh Scott T. R. , Cheng Hong , Bryson James W. , Roder Heinrich , DeGrado William F.
Published: July 2002

Although
de novo
protein design is an important endeavor with implications for understanding protein folding, until now, structures have been determined for only a few 25- to 30-residue designed miniproteins. Here, the NMR solution structure of a complex 73-residue three-helix bundle protein, α
3
D, is reported. The structure of α
3
D was not based on any natural protein, and yet it shows thermodynamic and spectroscopic properties typical of native proteins. A variety of features contribute to its unique structure, including electrostatics, the packing of a diverse set of hydrophobic side chains, and a loop that incorporates common capping motifs. Thus, it is now possible to design a complex protein with a well defined and predictable three-dimensional structure.
Adaptive evolution of color vision of the Comoran coelacanth (
Latimeria chalumnae
)
Authors: Yokoyama Shozo , Zhang Huan , Radlwimmer F. Bernhard , Blow Nathan S.
Published: July 2002

The coelacanth, a “living fossil,” lives near the coast of the Comoros archipelago in the Indian Ocean. Living at a depth of about 200 m, the Comoran coelacanth receives only a narrow range of light, at about 480 nm. To detect the entire range of “color” at this depth, the coelacanth appears to use only two closely related paralogous RH1 and RH2 visual pigments with the optimum light sensitivities (λmax) at 478 nm and 485 nm, respectively. The λmax values are shifted about 20 nm toward blue compared with those of the corresponding orthologous pigments. Mutagenesis experiments show that each of these coadapted changes is fully explained by two amino acid replacements.