DNA sequences required for the in vitro replication of adenovirus DNA.

Initiation of adenovirus (Ad) DNA replication occurs on viral DNA containing a 55-kilodalton (kDa) protein at the 5' terminus of each viral DNA strand and on plasmid DNAs containing the origin of Ad replication but lacking the 55-kDa terminal protein (TP). Initiation of replication proceeds via the synthesis of a covalent complex between an 80-kDa precursor to the TP (pTP) and the 5'-terminal deoxynucleotide, dCMP. Formation of the covalent pTP-dCMP initiation complex with Ad DNA as the template requires the viral-encoded pTP and DNA polymerase and, in the presence of the Ad DNA binding protein, is dependent upon a 47-kDa host protein, nuclear factor I. Initiation of replication with recombinant plasmid templates requires the aforementioned proteins and an additional host protein, factor pL. Deletion mutants of the Ad DNA replication origin contained within the 6.6-kilobase plasmid pLA1 were used to analyze the nucleotide sequences required for the formation and subsequent elongation of the pTP-dCMP initiation complex. The existence of two domains within the first 50 base pairs of the Ad genome, both of which are required for the efficient use of recombinant DNA molecules as templates in an in vitro DNA replication system, was demonstrated. The first domain, consisting of a 10-base-pair "core" sequence located at nucleotide positions 9-18, has been identified tentatively as a binding site for the pTP [ Rijinders , A. W. M., van Bergen, B. G. M., van der Vliet , P. C. & Sussenbach , J. S. (1983) Nucleic Acids Res. 11, 8777-8789]. The second domain, consisting of a 32-base-pair region spanning nucleotides 17-48, was shown to be essential for the binding of nuclear factor I.     

The influence of volume exclusion by chromatin on the time required to find specific DNA binding sites by diffusion

Within the nuclei of eukaryotic cells, the density of chromatin is nonuniform. We study the influence of this nonuniform density, which we derive from microscopic images [Schermelleh L, et al. (2008) Science 320:1332-1336], on the diffusion of proteins within the nucleus, under the hypothesis that chromatin density is proportional to an effective potential that tends to exclude the diffusing protein from regions of high chromatin density. The constant of proportionality, which we call the volume exclusivity of chromatin, is a model parameter that we can tune to study the influence of such volume exclusivity on the random time required for a diffusing particle to find its target. We consider randomly chosen binding sites located in regions of low (20th-30th percentile) chromatin density, and we compute the median time to find such a binding site by a protein that enters the nucleus at a randomly chosen nuclear pore. As the volume exclusivity of chromatin increases from zero, we find that the median time needed to reach the target binding site at first decreases to a minimum, and then increases again as the volume exclusivity of chromatin increases further. Random permutation of the voxel values of chromatin density abolishes the minimum, thus demonstrating that the speedup seen with increasing volume exclusivity at low to moderate volume exclusivity is dependent upon the spatial structure of chromatin within the nucleus.     

The time and change test: a simple screening test for dementia

BACKGROUND: Although dementia screening tests are available, they have not gained widespread use in hospital or primary care settings. Our goal was to develop a simple, standardized, performance-based test incorporating real-world activities to augment screening efforts in older populations: the Time and Change (T&C) Test. METHODS: The study followed a prospective cohort design, involving medicine and surgery services at an urban teaching hospital. From consecutive admissions, 776 participants aged 70-98 years, 14% with dementia, were enrolled. T&C ratings were validated against a reference standard based on the modified Blessed Dementia Rating Scale and the Mini-Mental State Examination (MMSE). Convergent validity with other cognitive measures, test-retest agreement, and interobserver reliability were assessed. RESULTS: The T&C Test had a sensitivity of 86%, specificity of 71%, and negative predictive value of 97%. The T&C Test demonstrated convergent agreement with three cognitive measures, agreeing most strongly with the MMSE (r = .58). Test-retest and interobserver agreement rates were 88% and 78%, respectively. Education explained 3% of the variance of the T&C Test, compared with 13% of the MMSE. The T&C Test took a mean of 22.9 seconds to complete and was acceptable to participants. Refusal of any test component occurred in 39 individuals (5%). CONCLUSIONS: The T&C Test is a simple, accurate, reliable, performance-based tool for detection of dementia. With its quick, easy-to-use, real-world nature, we hope the T&C Test will be used for widespread cognitive screening in older populations.     

Estimates for the pool size of releasable quanta at a single central synapse and for the time required to refill the pool.

Local superfusion of limited dendritic areas with hypertonic or hyperkalemic solutions stimulates the release of quanta from a small population of synapses made on rodent hippocampal neurons maintained in primary culture, and each quantal event can be detected in the postsynaptic neuron. With maintained stimulation, the initial release rate is about 20 quanta per sec per synapse, and this rate declines exponentially to a final low level. These observations can be interpreted as depletion of available quanta and, with this interpretation, a bouton would contain one to two dozen quanta in its readily releasable pool. Tests with a second application of the solution that produces release reveal that the pool of readily releasable quanta is replenished with a time constant of about 10 sec (36 degrees C). The pool of quanta defined in this way may correspond to the population of vesicles docked at the bouton's active zone.     

The DNA sequences required for apolipoprotein B expression in the heart are distinct from those required for expression in the intestine.

We recently reported that the apolipoprotein B (apo-B) gene is expressed in the heart and that the heart secretes apo-B-containing lipoproteins. We have also recently demonstrated that the tissue-specific expression of the apo-B gene is complex, with apo-B gene expression in the intestine being dependent on an enhancer element located very far from the structural gene (between 54 and 62 kb 5' to exon 1). In this study, we investigated whether these distant DNA sequences control or affect the expression of the apo-B gene in the heart. To address this issue, we analysed heart apo-B gene expression in heart tissue from a series of human apo-B transgenic mice generated from human genomic DNA constructs that ranged in size from 70 to 207 kb. All of these constructs contained the coding portion of the apo-B gene, but each contained different amounts of flanking sequences (5-120 kb 5' to the gene and 1.5-35 kb 3' to the gene). Each construct conferred human apo-B gene expression in the heart, including those containing as little as 5 kb of 5'-flanking sequences or as little as 1.5 kb of 3'-flanking sequences. These data support the idea that apo-B gene expression in the heart, unlike that in the intestine, is not affected by a distant enhancer element.     

recF and recR are required for the resumption of replication at DNA replication forks in Escherichia coli

Escherichia coli containing a mutation in recF are hypersensitive to UV. However, they exhibit normal levels of conjugational or transductional recombination unless the major pathway (recBC) is defective. This implies that the UV sensitivity of recF mutants is not due to a defect in recombination such as occurs during conjugation or transduction. Here, we show that when replication is disrupted, at least two genes in the recF pathway, recF and recR, are required for the resumption of replication at DNA replication forks, and that in their absence, localized degradation occurs at the replication forks. Our observations support a model in which recF and recR are required to reassemble a replication holoenzyme at the site of a DNA replication fork. These results, when taken together with previous literature, suggest that the UV hypersensitivity of recF cells is due to an inability to resume replication at disrupted replication forks rather than to a defect in recombination. Current biochemical and genetic data on the conditions under which recF-mediated recombination occurs suggest that the recombinational intermediate also may mimic the structure of a disrupted replication fork.     

The normal range and a simple diagram for recording whole gut transit time

The time taken for radio-opaque markers to pass through the intestine has been measured in 25 healthy men, and 18 healthy women in both the follicular and luteal phases of the menstrual cycle. The subjects collected all stools after ingestion of the markers, the number of markers present in each stool was counted on a radiograph, and the number of markers retained in the body was thus determined for 12 hourly intervals after ingestion. The mean values (2 standard deviations) for men and women in both phases of the menstrual cycle proved to be so similar that the results have been combined to provide a single normal range. These data for the normal range for retained markers (as assessed by plain radiograph) are presented in diagrammatic form for clinical use. To assess whether a patient's whole gut transit time lies within the normal range a single type of marker can be used and an abdominal radiograph performed at 12 or 120 hours, the limits of the normal range. Normal subjects retain more than 20% of markers within 12 hours and less than 80% after 120 hours. If desired more information can be gained by giving different types of marker on successive days, so that several transit studies providing intermediate values can be obtained from a single abdominal radiograph at 120 hours.

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Résumé Le temps mis par des marqueurs radio-opaques pour parcourir l'intestin a été mesuré chez 25 hommes sains et 18 femmes saines, à la fois dans les phases folliculiniques et lutéïniques du cycle menstruel. Les sujets collectaient toutes leurs selles après ingestion de marqueurs. Le nombre de marqueurs présents dans chaque selle était compté par radiographie, et le nombre de marqueurs retenus dans le corps était ainsi déterminé lors de douze intervalles horaires après ingestion. Les valeurs normales (±2 déviations standards) pour les hommes et les femmes dans les deux phases du cycle menstruel se sont révélées être tellement similaires que les résultats ont été combinés pour fournir, une valeur normale unique. La valeur normale des marqueurs retenus (affirmée sur la radiographie) est présentée sur un diagramme pour un usage clinique. Pour évaluer si le temps de transit intestinal d'un patient se tient dans des valeurs normales un simple type de marqueur peut être utilisé et une radiographie abdominale effectuée à 12 ou 120 h, les limites de valeurs normales. Les sujets normaux conservent plus de 20% des marqueurs en 12 h et moins de 80% après 120 h. Pour plus d'information on peut utiliser différents types de marqueurs à des jours successifs, ainsi plusieurs temps de transit fournissant des valeurs intermédiaires peuvent être obtenus d'une simple radiographie abdominale à 120 h.

     

A DNA gyrase-binding site at the center of the bacteriophage Mu genome is required for efficient replicative transposition.

We have discovered a centrally located site that is required for efficient replication of bacteriophage Mu DNA and identified it as a strong DNA gyrase-binding site. Incubation of Mu DNA with gyrase and enoxacin revealed a cleavage site 18.1 kilobases from the left end of the 37.2-kilobase genome. Two observations indicate a role for the site in Mu replication: mutants of Mu, able to grow on an Escherichia coli gyrB host that does not allow growth of wild-type Mu, were found to possess single-base changes resulting in more efficient gyrase binding and cleavage at the site. Introduction of a 147-base-pair deletion that eliminated the site from a prophage inhibited the onset of Mu replication for greater than 1 hr after induction.     

The nonhomologous end-joining pathway of DNA repair is required for genomic stability and the suppression of translocations

We have used spectral karyotyping to assess potential roles of three different components of the nonhomologous DNA end-joining pathway in the maintenance of genomic stability in mouse embryonic fibroblasts (MEFs). MEFs homozygous for mutations that inactivate either DNA ligase IV (Lig4) or Ku70 display dramatic genomic instability, even in the absence of exogenous DNA damaging agents. These aberrant events range from chromosomal fragmentation to nonreciprocal translocations that can involve several chromosomes. DNA-dependent protein kinase catalytic subunit deficiency also promotes genome instability. Deficiency for the p53 cell cycle checkpoint protein has little effect on spontaneous levels of chromosomal instability in Lig4-deficient fibroblasts. However, in the context of ionizing radiation treatment, p53 deficiency allowed visualization of massive acute chromosomal destruction in Lig4-deficient MEFs, which in surviving cells manifested as frequent nonreciprocal translocations. We conclude that nonhomologous DNA end-joining plays a crucial role as a caretaker of the mammalian genome, and that an alternative repair pathway exists that often leads to nonreciprocal translocations.     

DNA topoisomerase II mutant of Saccharomyces cerevisiae: topoisomerase II is required for segregation of daughter molecules at the termination of DNA replication.

A temperature-sensitive DNA topoisomerase II mutant of the yeast Saccharomyces cerevisiae has been identified. Genetic analysis shows that a single recessive nuclear mutation is responsible for both temperature-sensitive growth and enzymatic activity. Thus, topoisomerase II is essential for viability and the mutation is most probably in the structural gene. Experiments with synchronized mutant cells show that at the nonpermissive temperature cells can undergo one, and only one, round of DNA replication. These cells are arrested at medial nuclear division. Analysis of 2-microns plasmid DNA from these cells shows it to be in the form of multiply intertwined catenated dimers. The results suggest that DNA topoisomerase II is necessary for the segregation of chromosomes at the termination of DNA replication.     

DNA wrapping is required for DNA damage recognition in the Escherichia coli DNA nucleotide excision repair pathway

Localized DNA melting may provide a general strategy for recognition of the wide array of chemically and structurally diverse DNA lesions repaired by the nucleotide excision repair (NER) pathway. However, it is not clear what causes such DNA melting and how it is driven. Here, we show a DNA wrapping–melting model supported by results from dynamic monitoring of the key DNA–protein and protein–protein interactions involved in the early stages of the Escherichia coli NER process. Using an analytical technique involving capillary electrophoresis coupled with laser-induced fluorescence polarization, which combines a mobility shift assay with conformational analysis, we demonstrate that DNA wrapping around UvrB, mediated by UvrA, is an early event in the damage-recognition process during E. coli NER. DNA wrapping of UvrB was confirmed by Förster resonance energy transfer and fluorescence lifetime measurements. This wrapping did not occur with readily denaturable damaged DNA substrates (“bubble” DNA), suggesting that DNA wrapping of UvrB plays an important role in the induction of DNA melting around the damage site. Analysis of DNA wrapping of mutant UvrB Y96A further suggests that a cooperative interaction between DNA wrapping of UvrA₂B and contact of the β-hairpin of UvrB with the bulky damage moiety may be involved in the local DNA melting at the damage site.     

Mrc1 phosphorylation in response to DNA replication stress is required for Mec1 accumulation at the stalled fork

DNA replication stress activates a response pathway that stabilizes stalled forks and promotes the completion of replication. The budding yeast Mec1 sensor kinase, Mrc1 mediator, and Rad53 effector kinase are central to this signal transduction cascade in S phase. We report that Mec1-dependent, Rad53-independent phosphorylation of Mrc1 is required to establish a positive feedback loop that stabilizes Mec1 and the replisome at stalled forks. A structure–function analysis of Mrc1 also uncovered a central region required for proper mediator function and association with replisome components. Together these results reveal new insight into how Mrc1 facilitates checkpoint signal amplification at stalled replication forks.     

Upstream DNA sequences required for tissue-specific expression of the HLA-DR alpha gene

We have used in vitro deletion mutagenesis in combination with DNA transfection to search for cis-acting regulatory elements involved in the tissue-specific expression of a human class II major histocompatibility complex gene. A 140-base-pair 5' flanking fragment that contains the class II box consensus sequences and an octamer sequence (ATTTGCAT) confers tissue specificity on the promoter of the HLA-DR alpha gene. Recombinant DNA plasmids containing this DR alpha gene segment fused to the coding sequence of the bacterial chloramphenicol acetyltransferase gene are expressed at higher levels in human B-cell lines than in human T-cell lines. We have demonstrated that the most 5' of the class II boxes is essential for tissue-specific DR alpha promoter function. In addition, using an electrophoretic mobility shift assay to identify DNA binding proteins, we have detected binding of nuclear proteins to DNA probes containing the class II boxes and the octamer sequence. A protein that binds to the octamer is present at higher levels in nuclear extracts of B-cell lines than in other cell lines examined. This protein may be important for the tissue-specific expression of the HLA-DR alpha gene.     

A method for identifying the viral genes required for herpesvirus DNA replication.

Several laboratories have shown that transfected plasmid DNAs containing either of the two known origins of herpes simplex virus (HSV) DNA replication, oriS or oriL, are replicated in HSV-1-infected cells or in cells cotransfected with virion DNA. I have found that HSV-1 (KOS) DNA digested to completion with the restriction enzyme Xba I is as efficient as intact viral DNA in supporting the in vivo replication of cotransfected plasmids containing oriS. On the basis of this result, several of the Xba I restriction fragments of HSV-1 DNA were cloned into the plasmid vector pUC19, and combinations of cloned DNAs were tested for their ability to supply the trans-acting functions required for HSV origin-dependent replication. A combination of five cloned fragments of HSV-1 can supply all of the necessary functions: Xba I C (coordinates 0.074-0.294), Xba I F (coordinates 0.294-0.453), Xba I E (coordinates 0.453-0.641), Xba I D (coordinates 0.641-0.830), and EcoRI JK (coordinates 0.0-0.086; 0.830-0.865). Transient plasmid replication in this system is dependent on the presence of either oriS or oriL in cis. The plasmid containing Xba I F can be replaced by two smaller plasmids, one of which contains only the gene for the HSV-encoded DNA polymerase, and the other of which contains only the gene for the major DNA binding protein (ICP8). Thus, plasmid DNA replication in this system depends on two of the genes known from genetic studies to be essential for viral DNA replication in infected cells. This system defines a simple complementation assay for cloned fragments of HSV DNA that contain other genes involved in viral DNA replication and should lead to the rapid identification of all such genes.     

Age and simple reaction time: decade differences for 5,325 subjects

In a booth at a public exhibition entitled "Medicines for Man," 5,325 men, women, and children carried out a 1-minute test of simple reaction time (RT) with 1 to 10 second randomized variable preparatory interval (PI). They recorded their ages by decade. Average RT over the last eight (of ten) trials increased progressively from the 20s up to age 60 and over, and downward to the teens and under 10s. The single fastest RT in each test varied much less with age, only the 20s being clearly faster than the rest, with the under 10s slower. Within-subject variability of RT was increased only in the under 10s and over 60s. Ability to sustain attention during the longer PIs may underlie the gross average RT differences with age, and possibly some more basic neural property the superiority of the 20s in fastest RT.