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Hepatic adenoma (HA) and focal nodular hyperplasia (FNH) are two common non-malignant tumors of the liver. Genomic analysis on these benign lesions may shed light on the genetic mechanism underlying liver carcinogenesis. We used comparative genomic hybridization (CGH) to evaluate genomic changes in eight cases of HA and six cases of FNH, obtained by surgical procedures; the resulting chromosomal aberration profiles were analyzed together with their pathological and clinical manifestations. We found consistent chromosomal lesions associated with both non-malignant hepatic tumors. The overall genomic abnormalities in HA and FNH were much less obvious than those in hepatocellular carcinoma (HCC). Among these limited changes, frequent gains were located on chromosomal arms 1q (50%), 17q (50%), 1p (38%), and 11q (38%) in HA, and on 11q (50%), 9q (33%), 17q (33%), and 22q (33%) in FNH. Gains outnumbered losses, and HA contained more CGH abnormalities than did FNH. Interestingly, CGH alteration hotspots found in HA, but not in FNH, appeared largely to coincide with common genomic lesions of cancerous HCC, suggesting an interesting relationship along the tumorigenesis pathway of HA and HCC.

To investigate the chromosomal imbalances that occur in oral carcinoma associated primarily with betel use and their clinical implications, we performed chromosomal analysis using comparative genomic hybridization on 47 patients with this disease. The most common gains of chromosome arms were 8q, 9q and 11q, and the most frequent losses were of chromosomal arms 3p and 4q. The clinical parameters significantly associated with the numbers of chromosomal imbalances per tumor were the age of the patients and nodal metastasis. The preliminary findings of a lower incidence of loss of 4q and gain of 8q in betel-associated tumors compared to non-betel-associated tumors might provide insight into the carcinogenic effect of betel. Deletion of 3p and the gain of 11q alterations were more prevalent in carcinomas with lymph node metastasis than in node-negative tumors, indicating possible loci for metastasis suppressor or metastasis enhancing genes, respectively. Losses of 3p and 4q and gain of 9q were associated with poor outcome for the patients. These data demonstrated that the frequent aberrations in 4q and 9q sites can be used as novel prognostic predictors.

We analyzed genomic aberrations in 20 cervical adenocarcinomas by comparative genomic hybridization (CGH). Most tissue samples (85%) showed DNA copy number changes; gains were more common than losses. The most consistent region of chromosomal gain was mapped to chromosome arm 3q, found in 70% of the cases, with a minimal common region of 3q28-ter. Other recurrent amplifications of genetic material were detected on 17q (45%), 1p (30%), 1q (25%), and 11q (20%). High-level copy number increases were found in chromosomal regions 3q27-ter and 9pter-13. DNA losses were seldom observed, occurring primarily in underrepresented regions of chromosome arms 4q, 13q, and 18q. The presence of high-risk human papilloma virus genomes in the cervical adenocarcinoma samples was detected in 90% of the cases. However, there was no correlation between human papilloma virus type and the pattern of genomic changes. This study is the first report of CGH analysis in human cervical adenocarcinoma. Among the major genomic alterations, our results demonstrate the importance of DNA copy increases of chromosome arm 3q in the development of cervical adenocarcinoma and identify other amplified chromosomal regions that are also associated with cervical carcinogenesis.

Aberrant histone methylation contributes to tumor development and progression by histone methyltransferase. SETDB1 is an H3K9 methyltransferase that contributes to the epigenetic silencing of target genes in cancer cells, but the role of SETDB1 in the progression of tumorigenesis remains largely unknown. We show here that H3K9Me2 and SETDB1 are down-regulated in metastatic lung cancer cell lines. Immunohistochemistry examination of the lung carcinoma tissue microarray revealed that expression of SETDB1 is significant decreased in metastasis status. Ectopic expression of SETDB1 in highly invasive lung cancer cells suppressed cancer cell migration, invasion and filopodia formation. In contrast, knockdown of endogenous SETDB1 in non-invasive lung cancer cells by RNA interference increases cell migration and invasion in vitro. Mechanistic investigations suggested that SETDB1 is specifically down-regulated by miR-29b in metastatic lung cancer cells. Ectopic miR-29b expression in non-invasive lung cancer cells increases cell invasion ability. Furthermore, we also establish a causal role for the SETDB1/SMAD3 complex in driving metastasis and identify ANXA2 as a critical target in this process among numerous target genes. These findings suggest a novel pathway regulated by SETDB1/SMAD3 complex in lung cancer metastasis. Citation Format: Tsai-Yu Tzeng, Ming-Ta Hsu. A Novel pathway for regulation of ANXA2 expression in lung cancer cell metastasis by SETDB1/SMAD3 repressor complex. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2969. doi:10.1158/1538-7445.AM2013-2969

To study the mechanism by which ultraviolet (UV) light inhibits DNA replication, we examined the effects of UV 254 nm irradiation on the replication of simian virus 40 (SV40) DNA and SV40-based plasmid in monkey cells. The study was designed to determine the relative contributions made by inhibition of replication initiation and chain elongation to the immediate inhibition of DNA replication following UV irradiation. We used two-dimensional neutral-alkaline electrophoresis to examine the behaviour of replication intermediates unambiguously. Kinetic analysis using this technique showed that initiation of replication started to decline at 15 min post-irradiation. When the pulse label incorporated in SV40 replication intermediates before irradiation was chased for 1 h, most of the label was found in mature Form I and II molecules. This indicated that replication elongation took place on damaged template. We also used a transfection technique to show that heavily irradiated plasmids replicated efficiently in unirradiated transfected cells. By the transfection technique, we observed that UV irradiation of host cells dose-dependently inhibited replication of transfected non-irradiated plasmids, suggesting that the inhibition of DNA replication is due to a global change in cellular physiology induced by UV. This change was also apparent from poor staining of the chromatin by fluorescent-DNA-binding dyes immediately after UV irradiation of intact cells. We conclude that a significant fraction of chain elongation proceeds on damaged templates and DNA replication during the acute response of cells irradiated with UV is mainly controlled by the inhibition of replication initiation.

This paper presents the development of the light weight Phenolic Impregnated Carbon Ablators (PICA) and its thermal performance in a simulated heating environment for planetary entry probes. PICA material was developed as a member of the Light Weight Ceramic Ablators (LCAs) family, and since then, the manufacturing process of this material was significantly unproved. The density of PICA material ranges from 0.224 to 0.321 g/cc having uniform resin distribution within the fibrous substrate. Surface densification was also developed to improve the ablation characteristics of PICA against extremely high stagnation pressures. The thermal performance of PICA was evaluated in the Ames arc jet facility at cold wall heat fluxes from 425 to 3360 W/cm and surface pressures of 0.1 to 0.43 attn. Heat loads used in these tests varied from 6,245 to 33,600 J/cm and are representative of the entry conditions of several proposed Discovery missions. Surface and in-depth temperatures were measured by using optical pyrometers and thermocouples. Surface recession was also measured by using a template and a height gage. The ablation characteristics and efficiency of the PICA is quantified by using the effective heat of ablation, and the thermal penetration response is evaluated by the thermal soak data. In addition, comparison of the thermal performance of standard and surface densified PICA is also discussed.

New Light Weight Ceramic Ablators (LCAs) were produced by using ceramic and carbon fibrous substrates impregnated with silicone and phenolic resins. Special infiltration techniques (patent pending) were developed to control the amount of organic resin in the highly porous fiber matrices, so that the final densities of LCAs range from 0.224 to 0.30 g/cu cm. This paper presents the thermal and ablation performance of the Silicone Impregnated Reusable Ceramic Ablators (SIRCA) in a simulated Mars entry heating environment. Testing was conducted in the Ames 60 MW Interaction Heating Facility (IHF) and 20 MW Aerodynamic Heating Facility (AHF). Test results show that the ablation characteristics of SIRCA are divided into three regimes: non-receding, surface coalescent, and receding. Four different Reusable Surface Insulation (RSI) substrates were used in the production of SIRCA to determine the effect of substrate compositions on the ablation performance. SIRCA with high-purity silica fibers generally performed better at high heating rates. Samples of SIRCA were tested at heating rates ranging from 45 to 550 W/sq cm and at stagnation pressures of 0.02 to 0.47 atm. Several samples were also tested at a heating environment simulating the Mars Aerocapturing mission, which generally consists of a low heating rate and a very high heat load. Material characterizations were also conducted to evaluate the material's mechanical, thermal, and optical properties. The effective thermal conductivity of SIRCA is comparable to that of the RSI substrates, and the emissivity of the charred SIRCA was measured to be about 0.92. Several samples of SIRCA were also exposed to the same heating condition for five cycles, and no additional significant mass loss or recession was observed. (Author)

Existing methods of fetal lung organ culture are complicated and require special skills. With the use of a polyester-based plastic sheet, we have developed a simpler human fetal lung organ culture that is viable for 6 wk. This novel method permits the study of growth and differentiation, pulmonary surfactant secretion, and the response of human lung tissue to injury in vitro. Lung tissues, obtained from human fetuses ranging in gestational age from 14 to 18 wk, were cultured on the polyester sheet in Dulbecco's modified Eagle medium supplemented with 15% fetal calf serum and gentamicin. Microscopic study of the fetal lung before culturing revealed round epithelial tubules, lined by glycogen-rich columnar cells and a thick cellular interstitium. After 1 wk in culture, morphological examination showed the development and expansion of alveolar saccules and thinning of the interstitium; type I and II pneumocytes as well as fibroblasts and myofibroblasts were present. Lipid analysis of the tissues, 2 wk after the initiation of the culture, demonstrated a high percentage of dipalmitoyl phosphatidylcholine characteristic of pulmonary surfactant. Treatment of the organ culture with asbestos fibers induced type II cell hyperplasia, increased numbers of collagen fiber bundles within the interstitium, and the accumulation of multi-lamellated surfactant material within the alveolar lumens. We conclude that this organ culture system is suitable for studying lung growth, development, and injury in human tissue.

We investigated the in vivo effect of ellipticine, a mammalian topoisomeraseII(topoII) inhibitor, on SV40 DNA topology. In contrast to epipodophyllotoxins, ellipticine did not cause significant double stranded cleavage of intracellular SV40 DNA. Furthermore, ellipticine reduced cleavage induced by epipodophyllotoxins, VP16 and VM26. Unexpectedly, ellipticine dramatically increased the superhelical density of a fraction of intracellular SV40 DNA. Several lines of evidence suggest that the formation of this highly supercoiled DNA species (Ih form DNA) is not due to the inhibition of topoII per se, but is the result of intercalation by ellipticine in a subfraction of the intracellular SV40 chromatin followed by the fixation of DNA linking number by a topoisomerase activity. Based on the linking number change and the known unwinding angle of ellipticine, the intercalation density was calculated as one ellipticine molecule per 10-20 bp in the Ih DNA. This result suggests the existence of different populations of intracellular SV40 chromatin with respect to the accessibility to ellipticine intercalation.

Electron microscopic technique was used to examine the structures of SV40 DNA recombination intermediates induced by ultraviolet irradiation as an approach for understanding recombination mechanisms in animal cells. Putative recombination intermediate with the characteristic Holliday junction was observed in both SV40 and CV-1 monkey kidney cell DNA. These results suggest that Holliday recombination intermediate is a common intermediate in eukaryotic as well as prokaryotic recombination pathways. In UV irradiated cells, putative SV40 DNA recombination intermediates with multiple recombining partners were observed. In addition, UV irradiation induced two types of novel joint molecules of SV40 DNA. The first type contains replication intermediates as one of the joint molecules with the putative recombination junction located in the newly replicated DNA arms. The second type of novel joint molecules is represented by of the 'dumbbell' structures with two circular SV40 DNA linked by a linear DNA of varying lengths. The structures of these novel recombination intermediates suggest a strand-invasion mechanism for UV-induced DNA recombination.

The effects of adenovirus type 5 infection on the differentiation of cultured human skeletal myoblasts and of myoblast differentiation on the replication of adenovirus were investigated. Although infection of myoblasts concurrently with differentiation induction was inhibitory, myoblast differentiation was not impeded when infection was carried out 2 hr or later after induction. Similar studies conducted with EIA mutant viruses (d1312, pm975, and d11500) revealed that complete inhibition was dependent on the product of 13 S El A transcript expression, although partial inhibition could be induced by the 12 S product. Differentiation of myoblasts results in the generation of multinucleated myotubes and quiescent mononuclear cells. The three cell types (myoblasts, myotubes, mononuclear cells) were differentially permissive to adenovirus infection. The precursor myoblasts and the multinucleated myotubes were found to be permissive for adenovirus infection. The kinetics of their infection was delayed 24–48 hr relative to that of HeLa cells. Quiescent mononuclear cells in the differentiated myoblast cultures were found to be inefficient in supporting the production of adenovirus particles, despite the accumulation of adenovirus DNA and capsid proteins. Host protein synthesis in the three cell types also responded differently to adenovirus infection. In the multinucleated myotubes, host protein synthesis was potently inhibited by adenovirus at late times after infection, whereas it persisted in the proliferating myoblasts and quiescent mononuclear cells.

Single-stranded, linear DNA of simian virus 40 (SV40) created by denaturing the endonuclease EcoRI- or Hpa II-generated, linear, double-stranded products from form I DNA of SV40 was analyzed for regions of inverted repeated sequences by visualization with the electron microscope. Six hairpin loops were found at positions 0.11-0.30 (two loops forming a "rabbit ears" structure), 0.47-0.52, 0.63-0.68, 0.70-0.76, and 0.90-0.96. The nucleotide sequences within all of these inverted repeats may be related since the looped regions can crosshybridize with one another and, thus, the SV40 genome may contain regions of interspersed repeated and unique sequences. The map positions of the 3' and 5' ends of the early and late messenger RNAs, as determined by others, lie within regions of inverted repeated sequences. Previously recorded recombination events that occurred either within the SV40 genome or between SV40 DNA and other genomes have apparently occurred frequently at positions of inverted repeated sequences within the SV40 DNA.

Nucleoprotein complexes with in vitro transcription activity were isolated from HeLa cells late in lytic infection with Adenovirus type 2 (Ad2). Both polymerase II and polymerase III were active in these extracts, and greater than 85% of the labeled RNA was Ad2-specific. Electrophoretic analyses and Southern blot analyses demonstrated that RNA complementary to the entire 30 kb late transcription unit including RNA near the presumed termination site was synthesized. The addition of DRB (5,6,dichloro-1-beta-D-ribofuranosyl-benzimidazole) in vivo prior to the isolation of the complexes resulted in accumulation of polymerase II at the promoter proximal sites, but nascent chains started in vivo in DRB were successfully elongated in vitro. Approximately 10% of the RNA labeled in vitro contained poly(A), and the length of poly(A) was very similar to that of nuclear RNA isolated from Ad2-infected cells. The in vitro sites of poly(A) addition were specific--labeled poly(A)-terminated RNA molecules ended at a point on the genome coincident with previously mapped poly(A) sites of mRNAs produced in vivo. In addition, the polyadenylation enzyme (or enzymes) cosediment with the nucleoprotein complexes during sucrose gradient centrifugation since gradient purified complexes synthesize poly(A) containing RNA in vitro in the absence of any added nuclear extract.

An epoxy and carbon fiber composite has been used to produce a light-weight rocket case for the Space Shuttle. A kinetic model is developed which can predict the extent of epoxy conversion during the winding and curing of the case. The model accounts for both chemical and physical kinetics. In the model, chemical kinetics occur exclusively up to the time the transition temperature equals the reaction temperature. At this point the resin begins to solidify and the rate of this process limits the rate of epoxy conversion. A comparison of predicted and actual epoxy conversion is presented for isothermal and temperature programmed cure schedules.

Electron microscopic analysis of bis-psoralen crosslinked adenovirus type 5 virion DNA revealed supercoiled domains in an otherwise linear DNA. The existence of supercoiled arrangement in all the virion DNA was demonstrated by the sensitivity of Ad5 DNA in pentonless virus particles to the supercoiling-dependent endonucleolytic activity of Bal31 and S1 nucleases. These nucleases were found to cleave Ad5 virion DNA at specific sites. The observation of stable cleavage sites in the limit digestion of virion DNA by Bal31 suggests that cleavage sites represent boundaries of core proteins which impede the exonuclease activity of Bal31. These data suggest that specific arrangement of core proteins on Ad5 virion DNA. Based on this analysis we determined positions of core proteins in viral genome using indirect end labeling technique. The size of supercoiled domains of virion DNA was estimated by electron microscopy and also by boundaries of mutually exclusive Bal31 cleavage sites at limit digestion condition. Our data suggest each supercoiled domain is equal to about 12% of Ad5 genome length and about 8 loops can be accommodated in Ad5 virion. However sequences at two extreme ends of the viral genome were found to be outside of supercoiled domains. An interesting correlation between supercoiled domains and gene domains of Ad5 genome was noticed.

Molecules of adenovirus 2 DNA were isolated as nucleoprotein complexes late in infection of HeLa cells and examined by electron microscopy. Some were interpreted as representing intermediates in recombination events. The structures observed were consistent with predicted intermediates according to the model for genetic recombination proposed by Meselson and Radding [Meselson, M. S. & Radding, C. M. (1975) Proc. Natl. Acad. Sci. USA 72, 358-361] and constitute physical evidence from animal cells in support of this model.

The conformation of intracellular SV40 DNA during lytic infection of CV-1 cells was studied by the psoralen crosslinking technique. Analysis of the crosslinked SV40 DNA in the electron microscope revealed a rare population (0.1%) with a cruciform structure at coordinates 0.62 ± 0.05 or at 0.37 ± 0.05 of the SV40 genome. The implication of this observation in relation to SV40 DNA replication is discussed.

The nature of the G-loop heterogeneity and of the split end heterogeneity in Mu phage DNA have been studied. The structures of the molecules seen in self-renatured Mu phage DNA samples show that there are two classes of native molecules in which the G sequence occurs either in a direct or in an inverted order. By studying the structure of a single strand of Mu DNA when spread under weakly denaturing conditions, it is shown that the G region is flanked by very short (50 nucleotides or less) inverted repeat sequence. The G loop inversion is attributed to a phage enzyme-directed reciprocal recombination between these inverted repeat sequences. A second slightly larger inverted repeat sequence occurs within the G loop, but this sequence is not active in reciprocal recombination. By studying suitable diheteroduplexes of F-prime factors containing an inverted Mu prophage, with a reference F factor and with Mu phage DNA, it is shown that the highly heterogeneous split ends of Mu phage are lost when Mu becomes a prophage.

Partial denaturation pattern of sex factor deoxyribonucleic acid of Escherichia coli was studied by electron microscopy. Clustering of the adenine-plusthymine-rich regions in one part of the molecule was revealed. The positions of these regions were located on the physical map of F by analyzing the partial denaturation pattern of heteroduplexes between F and F-prime factors with various parts of F sequences deleted.

The sequence relations between some bacterial F-prime factors in Escherichia coli have been determined by observing, in the electron microscope, the pattern of duplex and single-stranded regions in heteroduplexes consisting of one strand from one episome and the complementary strand from another. All of the F-primes studied have a short piece of F missing. They appear to have been formed by excision from their respective Hfr's by a type I process (Scaife, 1967), leaving a piece of F remaining in the chromosome. The piece of F missing is the same in F lac and in the close relatives F450 and F 1s , although the former F-prime was derived from Hfr2 and the latter two from Hfr3. Thus, there is evidence for hot spots on F for insertion at different places in the Escherichia coli K12 chromosome to give Hfr's and hot spots for excision to give F-primes. However, a different sequence of F is missing in the episome F 8 . The physical structure of a number of F 8 episomes has been studied. In particular, we have studied the physical and genetic structures of a set of deleted F 8 episomes, prepared by P1 kc transduction of the original F 8 , and mostly deleted in some of the transfer genes. Thus, the various genetic markers have been physically mapped. A general model for the structure of F is proposed.

The reaction rate for the reduction of cyanocobalamin (B12) with Ti(III) in HCl and KCl solutions at constant ionic strength was followed by spectrophotometric measurements at 15°, 20°, 25° and 30°C. The reaction rats was found to be of pseudo-first-order with respect to B12 and of second-order with respect to B12 and Ti(III) in HCl solutions. The second-order rate constant was given by for the processes, The values of K0 obtained were 0.80∼1.29 liter mole−1 sec−1 in the range of ionic strength 0.2∼0.5 M at 30°. The average activation energy was 20.5 kcal mole−1.

Tandem repetition of domain in protein sequence occurs in all three domains of life. It creates protein diversity and adds functional complexity in organisms. In this work, we analyzed 52 streptococcal genomes and found 3748 proteins contained domain repeats. Proteins not harboring domain repeats are significantly enriched in cytoplasm, whereas proteins with domain repeats are significantly enriched in cytoplasmic membrane, cell wall and extracellular locations. Domain repetition occurs most frequently in S. pneumoniae and least in S. thermophilus and S. pyogenes. DUF1542 is the highest repeated domain in a single protein, followed by Rib, CW_binding_1, G5 and HemolysinCabind. 3D structures of 24 repeat-containing proteins were predicted to investigate the structural and functional effect of domain repetition. Several repeat-containing streptococcal cell surface proteins are known to be virulence-associated. Surface-associated tandem domain-containing proteins without experimental functional characterization may be potentially involved in the pathogenesis of streptococci and deserve further investigation.

The adenovirus E1B 19K gene plays an essential role in transformation of primary rodent cells in cooperation with E1A and in the inhibition of apoptosis during lytic infection. It has been shown that this E1B 19K protein is not necessary for viral DNA replication in human cell lines, such as HeLa and KB. We reported here that the E1B 19K mutant viruses were unable to replicate efficiently in a monocyte cell line, U937. Viral DNA synthesis and late gene expression were found to be defective in U937 cells infected with E1B 19K mutants compared with wild-type virus. Early viral RNA splicing patterns also differ between wild-type and dl 337-infected cells. Furthermore, the defect in viral replication could be complemented by dl 312 virus defective in E1A expression 4 days after infection with E1B mutants, suggesting persistence of the E1B mutant genome in the infected cells despite defective onset of the late phase of replication. These results imply that E1B 19K is required for efficient viral DNA replication in U937 cells. Inefficient DNA replication is also found in another monocyte cell line, THP-1.

Direct physical evidence confirming the Campbell model for the structure of prophage λ was obtained by observing, in the electron microscope, the structure of a heteroduplex between one strand of an episome, F450(λ ++ ), bearing a λ prophage, and the complementary strand of λ b 5 DNA. The facts that the resulting λ hybrid is circular and the concordance of the quantitative contour length measurements with expectation, both confirm the Campbell model. Additional confirmation plus the mapping of the att λ site on F450 are accomplished by constructing the diheteroduplex F450(λ + + )/F/λ b 2 b 5 c .

A recombinant DNA clone containing cellular sequences homologous to the transforming sequence, v-ros, of avian sarcoma virus UR2 was isolated from a chicken genomic DNA library. Heteroduplex mapping and nucleotide sequencing reveal that the v-ros sequences are distributed in nine exons ranging from 65 to 204 nucleotides on cellular ros (c-ros) DNA over a range of 11 kilobases. Comparison of the deduced amino acid sequences of c-ros and v-ros shows two differences: v-ros contains a three-amino-acid insertion within the hydrophobic domain presumed to be involved in membrane association, and (ii) the carboxyl 12 amino acids of v-ros are completely different from those of the deduced c-ros sequence. The deduced amino acid sequence of c-ros bears striking structural features similar to those of insulin and epidermal growth factor receptors, including the presumed hydrophobic membrane binding domain, amino acids flanking the domain, and the distance between the domain and the catalytic region of the kinase activity. The expression of c-ros appears to be under a very stringent control. When tissues at various stages of chicken development were analyzed, only kidney was found to contain a significant level of c-ros RNA. The level of c-ros RNA in kidney tissue is most abundant in 7- to 14-day-old chickens. Finally, nucleotide sequences of c-ros DNA and UR2-associated helper viral genome at regions corresponding to the gag ros recombination site suggest that the junction has been formed by RNA splicing.

For DNA in solution, there have been two methods for measuring the helical periodicity. One method examines the ladder of discrete bands formed in the electrophoretic pattern of supercoiled DNA. Such a ladder is assumed to reflect the set of differently linked covalently closed molecules. The other method measures the lengths of partial nuclease digestions of DNA segments affixed to flat surfaces. Neither method, however, is able to measure the absolute handedness. X-ray diffraction of DNA oligonucleotides can determine the handedness, but the DNA must necessarily be crystallized. We have developed a new method for determining the helical parameters of a segment of DNA in solution. It is the first non-crystallographic method that can directly determine both the helical periodicity and the absolute handedness of DNA. The results obtained using this method are consistent with the classical Watson-Crick right-handed double helical model.

The genetic and physical structures of commonly used F-prime factors carrying the galactose region of the Escherichia coli chromosome were analyzed. Deletions in the chromosomal DNA sequences in the F-prime factors were found to be frequent events. A genetic method was developed to reconstruct the original F-prime factors from deletion variants. Heteroduplex analysis of the reconstructed F-prime factors confirmed the derivation of the F-prime factors F100 and F152, from the same Hfr, and finally determined the normal E. coli chromosomal sequence in the region between fep and uvrB, containing about 5 min in genetic units and about 246.5 in kilobase units (kb). This sequence could be connected with the DNA sequences of the lac-purE region, which had been physically determined previously. Together they constituted a total of 528.6 kb. From these combined sequences, the distance from lacPO to galK was calculated to be 412.9 kb, which corresponds to 8.8 min in genetic units.

Electron microscopic examination of molecular hybrids between simian virus 40 DNA, that had been cleaved with EcoRI and then digested with exonuclease III, and either 16S or 19S mRNA produced late during the viral infection cycle indicated that each of these mRNAs contained a 5'-terminal "leader" sequence that was encoded in the viral genome at about map position 0.71-0.75. Hybridization of each of these mRNAs to viral DNA immobilized on nitrocellulose filters supported the electron microscopic observations.

Understanding the nature of DNA sequence differences among individuals is important to the understanding of fundamental questions in biology. To analyze such differences in complex genomes new approaches must be developed. We report two new techniques which aid in this effort. First, we have developed a modification of the Phenol Emulsion Reassociation Technique (PERT) that allows hybridization of long (20 kb and longer) single copy heteroduplex DNA fragments from human genomic DNAs. Secondly, by using a differential methylase protection technique we have shown that double methylase resistant heteroduplex DNA molecules can be size fractionated away from reannealed single methylase resistant homoduplex DNA molecules. These methods will be useful in obtaining DNA from chromosomal subregions linked to the inheritance of a specific trait or condition as described in the preceding paper and could also be used to create a map of the chromosomal subregion which includes the gene for the trait.

The question of whether or not 5′ leader sequences might confer characteristic translational properties to the mRNAs to which they are spliced was studied using two complementary experimental situations with respect to the composition of mRNA molecules. Late in adenovirus type 2 (Ad-2) infection of human cells, a common tripartite 5′ leader sequence is spliced to mRNA sequences coding for at least 13 different late viral proteins. This results in different mRNAs with identical 5′ leader sequences. In the second experimental system, identical coding sequences are spliced to different 5′ leader sequences with different caps. Wild-type SV40 VP1 mRNA (16 S) contains a 5′ leader sequence coded for at position 72–76 on the SV40 genome; in the SV40 mutant dl-808 part of the sequences in this region, including the sequence coding for cap, is deleted. The relative ability of these mRNAs to direct incorporation of radioactive amino acids into their respective polypeptide products was assayed in vivo under conditions of hypertonic initiation block (HIB) or in the presence of low concentrations of cycloheximide. Messenger RNAs with identical 5′ leader sequences were observed to exhibit very different responses in both experimental situations. Conversely, similar responses to HIB were observed for VP1 mRNAs with different 5′ leader sequences. It thus appears that the presence of specific sequences per se between the 5′ cap and the initiation codon does not alone confer characteristic initiation properties as measured indirectly by perturbation of the in vivo environment.

A new F-prime factor, F80, was isolated from an Escherichia coli strain harboring the F-prime factor F8 by selecting for transfer of the supE marker to a RecA- recipient. Genetic analysis shows that F80 carries a segment of the chromosomal DNA between lip and suc in addition to the tol-gal region normally in F8. Physical analysis by the electron microscope heteroduplex method suggests that the formation of F80 from F8 involves recombination between the alphabeta segment of F, which is present in F8, and the homologous sequence of F present in the E. coli chromosome at the site where F is supposed to integrate to form HfrP3. The implications of this result for the general mechanisms of F integration to form Hfr's are discussed.

In the presence of 100 mM Tris buffer (pH 7.5) and 1-10 mM Mg2+ EcoRI endonuclease cleaves DNA at a specific nucleotide sequence and in a characteristic way: -GAATTC-. But if Mg2+ is replaced by Mn2+, the specificity of the cleavage is relaxed and cleavages occur at many other sites; moreover, there appears to be a hierarchy of cleavage rates at the pseudo-EcoRI restriction sites. For example, SV40 DNA is cleaved only once in the usual digestion conditions, but with Mn2+ more than ten cleavages are made; the five most rapidly cleaved SV40 DNA map locations are 0/1.0 larger than 0.93 larger than 0.33 approximately equal to 0.42 larger than 0.29 approximately equal to 0.40 larger than 0.25. Mn2+ also alters the restriction specificity of HindIII but not HpaII endonuclease.

The study of potential regulatory events in gene expression in animal cells, such as RNA processing and template specificity for transcription or replication, was approached by visualization of these cellular processes in animal virus-infected cells. Viral nucleoprotein complexes were selectively extracted from purified HeLa cell nuclei 15 to 24 hours following infection with human adenovirus serotype 2. These viral nucleoprotein complexes contained replicative and transcriptional activities as judged by biochemical and structural analysis. Methods were developed that permitted electron microscopic visualization of adenovirus serotype 2 nucleoprotein complexes separated from the mass of cellular chromatin and containing measurable nascent RNA transcripts. Importantly, nascent RNA transcripts were observed on molecules that were intermediate in replication. Quantitation revealed that transcripts were present on replicating molecules with approximately the same frequency with which they were present on non-replicating molecules. This demonstrated that molecules of adenovirus serotype 2 DNA could serve simultaneously as templates for replication and transcription and that there was no obvious preference for replicating molecules as transcriptional templates.

The viral RNA segments in influenza virions were shown to be circular in conformation by using psoralen crosslinking methods. Electron microscopy of purified RNA following treatment of virus with the psoralen reagent 4'-aminomethyltrioxsalen (AMT) revealed circles with lengths corresponding to the individual segments. RNA blot analysis using polyacrylamide gels demonstrated that RNA from AMT-treated virus had a slowed migration, consistent with it being a single-stranded circle. Furthermore, nuclease S1 protection assays indicated that the termini of the RNA segments form an approximately 15-base-pair-long panhandle. This structure is consistent with the partial sequence complementarity that has been observed for the termini of all influenza virus RNAs. By RNA blot analysis, circular structures of viral sense RNA were also found in influenza virus-infected cells at early and late time points. The circular RNA was the predominant species at the time when the major transcription product is message RNA. This finding and the observation that the termination signal for mRNA synthesis directly abuts the panhandle suggest that a panhandle in the template viral RNA is a cis regulatory signal promoting the synthesis of mRNA instead of plus-sense template. Also, since the panhandle is present in high concentration in virions, we suggest that it is required for packaging and that the input RNA after infection is in the proper conformation for synthesis of primary transcripts.

The study of recombination in prokaryotes has been facilitated by the availability of recombinational mutants and simple genetic elements such as phages and plasmids. These small but defined molecules of DNA have been especially useful for electron microscopic analysis of structural detail of molecules undergoing recombination both in vivo and in vitro. A limitation in the structural analysis of plasmid recombination is the absolute number of recombining molecules which can be identified and analysed amidst a background of nonrecombining molecules. This limitation would be of even greater consequence in studies of genetic recombination in animal cells. We therefore chose virus-infected animal cells as a model system for the study of the molecular mechanism of genetic recombination in higher organisms. HeLa cells infected with adenovirus serotype 2 (Ad-2) offer several advantages for studying recombination: (1) the virus contains a small and well characterized genome of about 35 kilobases; (2) a large amount of Ad-2 DNA is accumulated during lytic infection and host DNA synthesis is suppressed; (3) Ad-2 recombines at a very high frequency; and (4) similar to phages, animal viruses and Ad-2 in particular are believed to use many of the host cell's enzymes in necessary metabolic processes, presumably including recombination. In this study we used electron microscopic techniques to visualize the structures of in vivo Ad-2 DNA recombination intermediates. Molecules were observed with structures at putative cross-over points which were consistent with the molecular mechanism of recombination proposed by Holliday. In addition, we observed Ad-2 DNA molecules engaged in recombination which were simultaneously serving as templates for replication and/or transcription. To the best of our knowledge, this is the first visualization of in vivo recombination intermediates of discrete DNA molecules isolated from eukaryotic cells.

It is shown, by electron microscope observation of the structures of heteroduplexes, that Mu-1 DNA inserted into the bacterial episomes F lac and F 8 [1] is collinear with, rather than a circulation permutation of, the DNA of the mature Mu-1 bacteriophage. Observation of the position of the inserted Mu defines a point within the gene that has been inactivated (the lacI gene for F lac and a transfer gene in F 8 [1] in these particular instances). These examples illustrate a new, general method for physical gene mapping. The episome with Mu DNA inserted into F 8 [1] [i.e., F 8 [1](Mu)], although derived from a single colony, is heterogeneous in that a self-renatured sample shows a nonhomology loop of length 3.0 kb. This nonhomology loop, which has previously been observed in mature Mu-1 DNA, is due to an inversion.