Increasing Pulse Length Research Articles

DNA replication intermediates in whole HeLa cells and isolated nuclei

Replicative intermediates have been studied in intact HeLa cells and in nuclei isolated from such cells. In whole cells the smallest DNA (primary DNA pieces) observed after pulse labelling with [ 3H]thymidine were 90–160 nucleotides long, and the size of the molecules in this class of DNA did not increase with increasing pulse length. Some increase in size was, however, observed when cells were pulse labelled at 25°C instead of 37°C. Chase experiments using nuclei from pulse-labelled cells suggested that the primary DNA pieces could be chased rapidly into DNA of high molecular weight (30–70 S, corresponding to a molecular weight of 0.7 · 10 7–6.4 · 10 7). Longer chases showed that the label eventually accumulated in DNA with s values > 150 S. In isolated nuclei the primary DNA pieces after a 1 min pulse at 37°C were approximately 200 nucleotides long. Primary pieces of this size were also rapidly chased into the 30–70 S region. However, during longer pulses in vitro a fraction of the primary DNA pieces grew beyond their normal size to reach a size of up to 2000–3000 nucleotides before being attached to the 30–70 S molecules.

Preswitching and postswitching phenomena in amorphous semiconducting films

Low-duty-cycle pulsed dc switching experiments have been performed on a variety of thin-film Te-based semiconducting glasses. No premonitory effects are observed in the current-time profile of the nonswitching off state. If, however, the threshold voltage is exceeded, a continuous current increase with time throughout the normal delay-time regime (preswitching off state) is always observed prior to switching. In a given device or film this current rise is interrupted, independent of overvoltage, by the rapid switching transition at about the same relative current increment above the current background extrapolated to zero time (leakage current). In the switched or on (filamentary conduction) state the current level, as limited by the series load resistor, can condition the response of the device in the subsequent preswitched off state. For loads in excess of ∼1 kΩ, there is no effect on the preswitching off state characteristics whereas, for appreciably smaller loads, there is a continuous increase in delay time with increased pulse length (time spent in the previous on state). The results (i) indicate that the development of a filamentary instability begins at the onset of the pulse and (ii) show that the operating threshold voltage can be raised (returned to its virgin value) when the operating current is large for a sufficiently long time. These features are consistent with switching models that treat the ``formed'' switching element as a high-conductance-formed filamentary region (of not necessarily uniform composition) surrounded by a low-conductivity glass.

Symmetrical in vivo transcription of mitochondrial DNA in HeLa cells.

RNA.DNA hybridization experiments utilizing separated strands of HeLa mitochondrial DNA and mit-RNA from HeLa cells exposed to short pulses of [5-(3)H]uridine have shown that the labeled RNA hybridizes with both the light (L) and the heavy (H) strand, though to a different relative extent depending upon the labeling time. Thus, hybridization of pulse-labeled RNA is about equal with the two strands when the pulse is very short (1-5 min), and becomes more and more predominant with the H strand with increasing pulse length. Pulse-labeled fast-sedimenting mit-RNA forms RNase-resistant double-stranded structures up to more than 5 mum long when self-annealed or annealed with an excess of unlabeled mit-RNA. These observations and the previous evidence of complete transcription of the H strand strongly suggest that mit-DNA is transcribed in HeLa cells symmetrically over a considerable portion of its length, with the transcript of the L strand being rapidly degraded or otherwise removed from the mitochondrial fraction.

Replication of bacteriophage M13: VI. Attachment of M13 DNA to a fast-sedimenting host cell component

During RF 3 3 Abbreviations used: RF, replicative form; RFI, the covalently closed double-stranded ring form; RFII, the double-stranded ring form containing one or more single-stranded breaks; SS, single strand; TdR, thymine deoxyriboside; dRFI, denatured RFI; PFU, plaque-forming unit. replication both parental and pulse labeled M13 DNA are associated with a fast sedimenting host cell component, tentatively identified as membrane. RF molecules containing parental label have equal amounts of linear and circular parental strands. Most of the pulse label in RF molecules is contained in strands of unit length or shorter. The proportion of pulse-labeled M13 DNA associated with membrane decreases with increasing pulse lengths. The freely sedimenting M13 DNA components are identified as RFI and RFII. Pulse label is found in both strands of membrane bound RFII, but in only the viral strand of freely sedimenting RFII. M13 DNA pulse labeled during single-strand synthesis also appears to be membrane associated, the proportion decreasing with increasing pulse lengths. The pulse label is contained exclusively in the viral strand of the bound RF molecules.

Replication of bacteriophage M13: II. The role of replicative forms in single-strand synthesis

Sucrose-gradient sedimentation of DNA from cells pulse-labeled with tritiated thymidine at 130 minutes after M13 infection shows a flow of material from RF ‡ ‡ Abbreviations used: RF, replicative form; RFI, the covalently closed double-stranded ring form; RFII, the double-stranded ring form containing one or more single-strand breaks; SS, single strand; TdR, thymine deoxyriboside. to progeny single strands. For pulses less than 40 seconds, most of the phagospecific DNA sediments at the rates of RFI and RFII. Pulses for 15 seconds or less show a third component sedimenting even more rapidly than RFI. All of the pulse label in this component is contained in linear viral strands, some of which sediment even more rapidly than circular viral DNA in alkaline sucrose gradients. The label in RFII is contained almost entirely in linear viral strands of unit length. The relative amount of label in RFII decreases with increasing pulse lengths up to about 180 seconds. After a lag of at least 40 seconds, label rapidly accumulates in progeny single strands; by five minutes the distribution of label is similar to that observed in long-term labeling with 70 to 80% of the label in single-stranded DNA. When pulse-labeling is followed by growth in unlabeled medium, most of the incorporated label is chased into progeny single strands. Label is found in both viral and complementary strands of RF only after long-term labeling from the time of infection. Labeled complementary strands of RF molecules are conserved during further growth in unlabeled medium while labeled viral strands are displaced from the RF as new viral strands are synthesized.

Monostatic Scattering from the Ocean Volume

Monostatic volume-scattering experiments were conducted in the Atlantic with a piston transducer operated at 19.5 kc/sec and having a beam width of approximately 8° at the half-power points. This investigation was made to determine the correspondence between measured returns and a model based on the theory of isotropic scattering from a volume. Intensities from measured values were averaged and the volume-scattering strength computed as a function of depth for several depression angles and for a range of pulse lengths. Scattering strength was found to be independent of the ensonified volume in regions of uniform scattering strength. In regions of varying scattering strength, increasing pulse length or volume tended to smooth the variations in scattering strength.