Oculopharyngeal muscular dystrophy in Hispanic New Mexicans.

CONTEXT: Oculopharyngeal muscular dystrophy (OPMD) is a rare myopathy caused by polyalanine triplet repeat expansion in the gene for poly(A) binding protein 2 (PABP2) and is found in isolated cohorts throughout the world. We have observed numerous cases of OPMD in New Mexico. OBJECTIVE: To characterize the clinical, genetic, and demographic features of the OPMD population in New Mexico. DESIGN, SETTING, AND PARTICIPANTS: Cohort study with analysis of outpatient clinic medical records from 1965 to 2001 at the University of New Mexico Hospital and the New Mexico VA Health Care System in Albuquerque, which serve the entire state. MAIN OUTCOME MEASURES: Clinical phenotype, supplemented with genetic confirmation (n = 10 patients) and in-depth clinical evaluations (n = 49 patients). RESULTS: We identified 216 cases of OPMD (99 women and 117 men) from 39 kindreds of New Mexicans spanning up to 4 generations. All patients were Hispanic, and the majority of probands came from northern New Mexico. In patients who had both ocular and pharyngeal muscle weakness, ptosis was just as likely to occur before or concurrent with dysphagia. Proximal limb muscle weakness and gait abnormalities were common and occurred later than ocular or pharyngeal weakness. The clinical expression of OPMD caused marked debility, although life-table analysis showed no decrease in life expectancy compared with unaffected family members (P =.81). Ten individuals from different kindreds were found to have an identical polyalanine triplet repeat expansion ([GCG](9)) in the PABP2 gene. CONCLUSIONS: Individuals in this cohort had clinical and genetic characteristics of classic OPMD. Longevity was not affected, but patients experienced considerable morbidity. The origin of the PABP2 mutation in New Mexican OPMD patients is unclear, although the geographic and genetic isolation of northern New Mexicans with a long ancestry in this region may have contributed to the development of this cohort. This disease cohort represents a large and previously unrecognized health care issue in the state of New Mexico and should serve to raise the awareness of this disorder among clinicians who treat Hispanics in the Southwest and throughout the United States.

Intranuclear inclusions in oculopharyngeal muscular dystrophy contain poly(A) binding protein 2.

Intranuclear inclusions are one of the ultrastructural hallmarks of oculopharyngeal muscular dystrophy (OPMD), a disorder caused by small polyalanine (GCG) expansions in the gene that codes for a ubiquitous nuclear protein called poly(A) binding protein 2 (PABP2). We studied OPMD skeletal muscle and found that 1.0 to 10.0% of myocyte nuclei contained discreet PABP2 immunoreactive intranuclear inclusions, providing the first direct evidence of the relation between the proposed gene for OPMD and the pathology of OPMD.

The nuclear poly(A) binding protein, PABP2, forms an oligomeric particle covering the length of the poly(A) tail.

The mammalian nuclear poly(A) binding protein, PABP2, controls the length of the newly synthesized poly(A) tail on messenger RNAs. To gain a better understanding of the mechanism of length control, we have investigated the structure of the PABP2.poly(A) complex. Electron microscopy and scanning force microscopy studies reveal that PABP2, when bound to poly(A), forms both linear filaments and discrete-sized, compact, oligomeric particles. The maximum diameter of the filament is 7 nm; the maximum diameter of the particle is 21(+/-2) nm. Maximum particle size is realized when the PABP2. poly(A) complex is formed with poly(A) molecules 200-300 nt long, which corresponds to the average length of the newly synthesized poly(A) tail in vitro and in vivo. The equilibrium between filaments and particles is highly sensitive to ionic strength; filaments are favored at low ionic strength, while particles predominate at moderate to high ionic strength. Nitrocellulose filter binding and gel mobility shift assays indicate that the PABP2.poly(A) particle formed on A(300) is not significantly more stable than complexes formed with smaller species of poly(A). These results are discussed in the context of the proposed functions for PABP2.

Intramolecular synapsis of duplex DNA by vaccinia topoisomerase.

Complexes formed by vaccinia topoisomerase I on plasmid DNA were visualized by electron microscopy. The enzyme formed intramolecular loop structures in which non-contiguous DNA segments were synapsed within filamentous protein stems. At high enzyme concentrations the DNA appeared to be zipped up within the protein filaments such that the duplex was folded back on itself. Formation of loops and filaments was also observed with an active site mutant, Topo-Phe274. Binding of Topo-Phe274 to relaxed DNA circles in solution introduced torsional strain, which, after relaxation by catalytic amounts of wild-type topo-isomerase, resulted in acquisition of negative supercoils. We surmise that the topoisomerase-DNA complex is a plectonemic supercoil in which the two duplexes encompassed by the protein filaments are interwound in a right handed helix. We suggest that topoisomerase-mediated DNA synapsis plays a role in viral recombination and in packaging of the 200 kbp vaccinia genome during virus assembly.

DNA looping by Ku and the DNA-dependent protein kinase.

The DNA-dependent protein kinase (DNA-PK) is required for DNA double-strand break (DSB) repair and immunoglobulin gene rearrangement and may play a role in the regulation of transcription. The DNA-PK holoenzyme is composed of three polypeptide subunits: the DNA binding Ku70/86 heterodimer and an approximately 460-kDa catalytic subunit (DNA-PKcs). DNA-PK has been hypothesized to assemble at DNA DSBs and play structural as well as signal transduction roles in DSB repair. Recent advances in atomic force microscopy (AFM) have resulted in a technology capable of producing high resolution images of native protein and protein-nucleic acid complexes without staining or metal coating. The AFM provides a rapid and direct means of probing the protein-nucleic acid interactions responsible for DNA repair and genetic regulation. Here we have employed AFM as well as electron microscopy to visualize Ku and DNA-PK in association with DNA. A significant number of DNA molecules formed loops in the presence of Ku. DNA looping appeared to be sequence-independent and unaffected by the presence of DNA-PKcs. Gel filtration of Ku in the absence and the presence of DNA indicates that Ku does not form nonspecific aggregates. We conclude that, when bound to DNA, Ku is capable of self-association. These findings suggest that Ku binding at DNA DSBs will result in Ku self-association and a physical tethering of the broken DNA strands.

TRAP, the trp RNA-binding attenuation protein of Bacillus subtilis, is a toroid-shaped molecule that binds transcripts containing GAG or UAG repeats separated by two nucleotides.

The trp RNA-binding attenuation protein of Bacillus subtilis, TRAP, regulates both transcription and translation by binding to specific transcript sequences. The optimal transcript sequences required for TRAP binding were determined by measuring complex formation between purified TRAP protein and synthetic RNAs. RNAs were tested that contained repeats of different trinucleotide sequences, with differing spacing between the repeats. A transcript containing GAG repeats separated by two-nucleotide spacers was bound most tightly. In addition, transmission electron microscopy was used to examine the structure of TRAP and the TRAP-transcript complex. TRAP was observed to be a toroid-shaped oligomer when free or when bound to either a natural or a synthetic RNA.

Snapshot blotting: transfer of nucleic acids and nucleoprotein complexes from electrophoresis gels to grids for electron microscopy.

We present a technique, "snapshot blotting," for the electrophoretic transfer of nucleic acids and nucleoprotein complexes in gel electrophoresis bands onto highly stable carbon film-coated grids for imaging by electron microscopy. The method permits structural analysis of macromolecular species that have been resolved by a gel mobility-shift assay. To demonstrate the efficiency and integrity of the transfer process for a multiprotein-DNA assembly, we have imaged various species of a prokaryotic transcription complex, using the cleavage-defective EcoRI(Q111) protein as an orientation marker and as a blockade of transcription elongation. Snapshot blotting should be of great utility in the structural characterization of nucleic acids and protein-nucleic acid interactions.

3'-end formation at the phage lambda tR1 rho-dependent transcription termination site.

The rho-dependent transcription terminator tR1 of bacteriophage lambda stops RNA synthesis downstream of the major rightward promoter, PR, shortly after the cro gene. Terminated transcripts produced in a purified in vitro transcription system display a heterodisperse set of 3' termini, occurring in clusters located at +290-300, 308-312, 340-345, 385-390, and 440-450 nucleotides from the transcription start site [Morgan, W.D., Bear, D.G., & von Hippel, P.H. (1983) J. Biol. Chem. 258, 9553-9564]. However, transcripts from the same promoter in vivo have been reported to end primarily at +310-312 [Court, D., Brady, C., Rosenberg, M., Wulff, D. L., Behr, M., Mahoney, M., & Izumi, S. (1980) J. Mol. Biol. 138, 231-254]. In order to understand the nature of this discrepancy, we have carried out a comparative analysis of lambda PR transcription products produced in translationally active S30 cell extracts, in a purified in vitro system and in vivo. RNAs from the cell extracts coupled to translation show primarily three PR-derived transcripts beginning at one predominant 5' end and terminating at +263, 308, and 318. Sites +263 and +308 appear to be RNA processing sites. S1 nuclease mapping studies of RNAs produced in vivo show one 5' end and two 3' termini ending at +263 and 311; the +263 site is the predominant 3' end. When transcripts produced in a purified in vitro transcription system are incubated in the S30 cell extract under various conditions, the RNAs are degraded to two primary products with lengths of 263 and 308-311 nt.(ABSTRACT TRUNCATED AT 250 WORDS)

Escherichia coli helicase II (UvrD) protein initiates DNA unwinding at nicks and blunt ends.

The Escherichia coli uvrD gene product, helicase II, is required for both methyl-directed mismatch and uvrABC excision repair and is believed to function by unwinding duplex DNA. Initiation of unwinding may occur specifically at either a mismatch or a nick, although no direct evidence for this has previously been reported. It has recently been shown that helicase II can unwind fully duplex linear and nicked circular DNA with lengths of at least approximately 2700 base pairs in vitro; hence, a flanking region of single-stranded DNA is not required to initiate DNA unwinding. In studies with uniquely nicked duplex DNA, we present EM evidence that helicase II protein initiates DNA unwinding at the nick, with unwinding proceeding bidirectionally. We also show that helicase II protein initiates DNA unwinding at the blunt ends of linear DNA, rather than in internal regions. These data provide direct evidence that helicase II protein can initiate unwinding of duplex DNA at a nick, in the absence of auxiliary proteins. We propose that helicase II may initiate unwinding from a nick in a number of DNA repair processes.

Stable DNA heteroduplex formation catalyzed by the Escherichia coli RecA protein in the absence of ATP hydrolysis.

A question remaining to be answered about RecA protein function concerns the role of ATP hydrolysis during the DNA-strand-exchange reaction. In this paper we describe the formation of joint molecules in the absence of ATP hydrolysis, using adenosine 5'-[gamma-thio]triphosphate (ATP[gamma S]) as nucleotide cofactor. Upon the addition of double-stranded DNA, the ATP[gamma S]-RecA protein-single-stranded DNA presynaptic complexes can form homologously paired molecules that are stable after deproteinization. Formation of these joint molecules requires both homology and a free homologous end, suggesting that they are plectonemic in nature. This reaction is very sensitive to magnesium ion concentration, with a maximum rate and extent observed at 4-5 mM magnesium acetate. Under these conditions, the average length of heteroduplex DNA within the joint molecules is 2.4-3.4 kilobase pairs. Thus, RecA protein can form extensive regions of heteroduplex DNA in the presence of ATP[gamma S], suggesting that homologous pairing and the exchange of the DNA molecules can occur without ATP hydrolysis. A model for the RecA protein-catalyzed DNA-strand-exchange reaction that incorporates these results and its relevance to the mechanisms of eukaryotic recombinases are presented.

Imaging of metal-coated biological samples by scanning tunneling microscopy.

A method for imaging biological samples by scanning tunneling microscopy (STM) is presented. There are two main difficulties in imaging biological samples by STM: (1) the low conductivity of biological material and (2) finding a method of reliably depositing the sample on a flat conducting surface. The first of these difficulties was solved by coating the samples with a thin film of platinum-carbon. The deposition problem was solved by a method similar to a procedure used to deposit biological molecules onto field ion microscope (FIM) tips. STM images of bacteriophage T7 and filamentous phage fd are shown. The substrate on which the samples were absorbed was atomically flat gold. The images do not show molecular detail due to the metal coating, but the gross dimensions and morphology are correct for each type of virus. Also, the surface density of virus particles increases and decreases in the way expected when the conditions of deposition are changed. These methods allow reliable and reproducible STM imaging of biological samples.

A plasmid vector and quantitative techniques for the study of transcription termination in Escherichia coli using bacterial luciferase.

We have developed a plasmid expression vector for the study of transcription terminators in Escherichia coli that utilizes the lux genes coding for the enzyme luciferase of the bioluminescent marine bacterium, Vibrio harveyi. The pBR322-derived plasmid, called pHV100, contains the E. coli lac promoter, the polylinker regions from the plasmid vector pUC18, and the V. harveyi lux genes. Insertion of transcription termination sites into the polylinker region results in decreased luciferase expression. Because the bioluminescence genes are not indigenous to E. coli, their expression can be studied in virtually any host strain without the complications of background activity. This facilitates sensitive measurements of terminator efficiency in hosts containing termination factor mutations. Bioluminescence can be easily monitored with high sensitivity, using a rapid photographic technique or a more quantitative photometric assay.

Interactions of Escherichia coli transcription termination factor rho with RNA. I. Binding stoichiometries and free energies.

In this paper we examine the binding of Escherichia coli transcription termination factor rho to single-stranded RNA. Random polyribonucleotide copolymers containing low ratios of the fluorescent base 1,N6-ethenoadenosine have been synthesized using polynucleotide phosphorylase. Binding of rho to these polynucleotides elicits a significant increase in fluorescence, thus allowing either the direct monitoring of the titration of these polynucleotides with rho or measurement of the competitive displacement of the protein from these probes with other nucleic acids, even in the presence of biologically significant concentrations of ATP. By these techniques, it is shown that the binding site size (n) of rho protein to polynucleotides is 13(+/- 1) nucleotide residues per rho monomer (or 78(+/- 6) nucleotide residues per rho hexamer). Binding constants (K) and co-operativity parameters (omega) for the binding of rho to these polynucleotides have been measured as a function of nucleotide composition and of salt concentration. The results show that the affinity of rho for cytosine residues is quite strong and salt concentration independent, whilst binding to uridine residues is somewhat weaker and very salt concentration dependent. Poly(rC) and poly(dC) bind to rho competitively and with equal affinity and site size, although poly(rC) is the strongest cofactor for activating rho-dependent ATPase and poly(dC) has no ATPase cofactor activity at all. It is also shown that ATP (or ADP or ATP-gamma-S) binding does not change the binding site size of rho on RNA nor decrease its affinity for RNA binding. Circular dichroism measurements of rho binding to phage R17 RNA suggest that the affinity (K omega) of rho for RNA may be increased by ATP. The possible significance of these results for models of rho-dependent transcription termination is discussed in the companion paper.

Interactions of Escherichia coli transcription termination factor rho with RNA. II. Electron microscopy and nuclease protection experiments.

Structural aspects of the interaction between Escherichia coli transcription termination factor rho and RNA have been investigated, using nuclease protection assays and electron microscopy. A synthetic RNA, poly(rC), has been used as a substrate for these studies, since it binds tightly to rho and acts as a strong activator of the ATPase activity of rho. Digestion of oligo(rC)-rho complexes with ribonuclease A yields oligo(rC) fragments with a maximum length of 70 to 80 nucleotide residues. Electron micrographs demonstrate that rho binds to poly(rC) as a toroid-shaped oligomer with an outside diameter of approximately 120 A. Taken together with data from the accompanying paper, which shows that the RNA binding site size per rho monomer is 13(+/- 1) nucleotide residues, we infer that rho binds RNA as a hexamer with an oligomeric site size of 72 to 84 residues. Further analysis of the electron micrographs has revealed that the polynucleotide chain is wrapped around, or condensed within, the protein oligomer. rho hexamers bind to poly(rC) with moderate co-operativity (omega = 380 +/- 60), displaying no significant preference for binding to chain ends versus internal sites on the polynucleotide chain. These findings and those of the companion paper are discussed in terms of various models for the structure of the rho-RNA complex in transcription termination.

RNA sequence and secondary structure requirements for rho-dependent transcription termination.

The interaction of E. coli termination factor rho with the nascent RNA transcript appears to be a central feature of the rho-dependent transcription termination process. Based on in vitro studies of the rho-dependent termination of the transcript initiated at the PR promoter of bacteriophage lambda, and on earlier studies, Morgan, Bear and von Hippel (J. Biol. Chem. 258, 9565-9574, 1983) proposed a model defining the features of a potential binding site for rho protein on transcripts subject to rho-dependent termination. This model suggested that an effective rho binding site on a nascent RNA transcript should be: (i) greater than 70-80 nucleotide residues in length; (ii) essentially unencumbered with stable secondary structure; (iii) relatively sequence non-specific; and (iv) located within a few hundred nucleotide residues upstream of the potential rho-dependent terminus. In this paper we examine the sequences and secondary structures of several transcripts that exhibit rho-dependent termination to test this hypothesis further. Unstructured regions of approximately the expected size and location were found on all the transcripts examined. Though several short specific sequence elements were found to occur in a very similar arrangement on the lambda PR- and lambda PL-initiated transcripts of lambda phage, no such elements of sequence regularity were found on any of the other rho-dependent transcripts. The results of the sequence comparisons reported here strongly support the generality of the "unstructured binding site" hypothesis for rho-dependent termination.

Escherichia coli transcription termination factor rho has a two-domain structure in its activated form.

Limited tryptic digestion of Escherichia coli transcription termination factor rho [an RNA-dependent nucleoside triphosphatase (NTPase)] yields predominantly two fragments (f1 and f2) when the protein is bound to both poly(C) and ATP. The apparent molecular masses of the two fragments are 31 kDa for f1 and 15 kDa for f2, adding up to the molecular mass of the intact rho polypeptide chain (46 kDa). Sequence analysis of the amino termini demonstrates that f1 is derived from the amino-terminal portion of rho and that the trypsin cleavage that defines f2 occurs at lysine-283. These results suggest that, in the liganded (activated) form, the native rho protein monomer is organized into two distinct structural domains that are separable by a single proteolytic cleavage. The f1 fragment, purified from NaDodSO4/polyacrylamide gels and renatured, binds poly(C) but the f2 fragment does not; neither regains any ATPase activity. ATP- and polynucleotide-dependent changes in the rate of proteolysis and in the character of the fragments produced suggest that rho undergoes a series of conformational transitions as a consequence of RNA binding, NTP binding and NTP hydrolysis. The rate of loss of rho ATPase activity and of intact rho monomers is slower in the presence of adenosine 5'-[gamma-thio]triphosphate than in the presence of either ATP or ADP, indicating that the hydrolysis of ATP may result in different conformational effects than does the binding of this ligand. These findings are discussed within the context of recent models of rho-dependent transcription termination.

Specificity of release by Escherichia coli transcription termination factor rho of nascent mRNA transcripts initiated at the lambda PR.

We have studied the specificity and kinetics of release of nascent RNA from ternary transcription complexes by Escherichia coli transcription termination factor rho in vitro. Stable ternary complexes, initiated at the lambda PR promoter, were prepared either by quenching the elongation reaction with EDTA or by preventing further elongation by incorporating 3'-O-methyl nucleotides at the 3' end of the nascent RNA chains. We find that rho protein can only release lambda PR-initiated transcripts from ternary complexes in which transcription has proceeded beyond 288 base pairs from PR; shorter chains are not released. Substitution of inosine for guanosine in the nascent RNA permits the rho-dependent release process to operate on complexes located as close as 108-116 base pairs downstream from PR. The regions of the template from which rho can release transcripts correspond, for both guanosine- and inosine-containing RNA, to those within which rho-dependent termination has also been shown to occur (Morgan, W. D., Bear, D. G., and von Hippel, P. H. (1983) J. Biol. Chem. 258, 9553-9564, 9565-9574). The half-time for the major part of the release process is less than 10 s. These results are in good accord with the hypothesis that the specificity of rho-dependent termination is jointly determined by two separable processes: (i) the specificity of rho binding to the nascent RNA chain and (ii) the location and strength of RNA polymerase-pausing sites.

Rho-dependent termination of transcription. I. Identification and characterization of termination sites for transcription from the bacteriophage lambda PR promoter.

We have conducted a detailed investigation of in vitro transcription from the bacteriophage lambda PR promoter in order to examine various aspects of the mechanism of rho-dependent termination. In these studies, we have focused particularly on nucleotide sequence specificity, both at the termini and at potential rho-binding sites on the mRNA, and on the relationships between elongation, pausing, and termination. Rho-terminated transcripts from restriction fragment templates have been analyzed by polyacrylamide gel electrophoresis, and termination efficiencies have been established by densitometry of autoradiographs. Termination sites on the template have been located by comparing the electrophoretic mobilities of terminated transcripts with those of transcripts of known length that have been artificially terminated by the incorporation of 3'-O-methyl nucleotides. We have identified five discrete rho-dependent termination sites located between 290 and 450 base pairs downstream from the lambda PR promoter. These rho-dependent 3'-termini are somewhat heterogeneous in details of sequence and potential RNA secondary structure, but all possess features that appear to be characteristic of RNA polymerase elongation pausing sites (Morgan, W. D., Bear, D. G., and von Hippel, P. H. (1983) J. Biol. Chem. 258, 9565-9574). The efficiency of termination at individual sites ranges from 20 to 70% under the usual in vitro transcription conditions; termination is inhibited by increasing the monovalent salt concentration. Lowering nucleoside triphosphate substrate concentrations increases termination efficiency at some sites located 290 or more base pairs downstream from PR, but does not enhance termination at sites closer to PR. The substitution of inosine for guanosine residues in the transcript, which decreases the stability of the RNA-DNA hybrid and of secondary structure in the nascent mRNA, results in strong rho-dependent termination at several new sites located 100 to 260 base pairs downstream from PR. In Morgan et al. (cited above), data on RNA polymerase elongation pausing as a function of reaction conditions are correlated with these termination results, and a general model for rho-dependent termination is discussed.