Chromosome duplication (DNA synthesis) was studied in cultured cells of Chinese hamsters by means of autoradiography following thymidine-H a incorporation. The technique used was to expose an asynchronously dividing population of rapidly growing cells for a 10 minute interval to a medium with thymidlne-H a. Cells were then transferred to a medium with excess unlabeled thymidine. The population was sampled at intervals thereafter and studies made of the frequency of labeled interphases and division figures, and the patterns of labeling of specific chromosomes,The average generation time during these experiments was about 14 hours.

CHO cells in culture were synchronized by mitotic selection, allowed to reattach to plastic flasks, and reach S phase in the presence of fluorode-oxyuridine at concentrations known to completely block the synthesis of thymidylate. The cells were released from the block with 3H-thymidine for pulses of 4, 8, 12, 24 and 40 min and DNA fiber autoradiographs prepared. An analysis of the spacing between origins of replication indicates that sites are available at intervals of about 4 mum along most of the DNA. Chain growth proceeds at about 1,000 nucleotides per minute and some of the closely situated sites become continuous, labeled segments after 8-12 min. However, unlabeled segments are still present between the replicated segments after 40 min. The data may be interpreted as evidence for regularly spaced initiation sites which are available in CHO cells, even though only one in 10-15 of these may be utilized for initiation each cycle under normal growth conditions in these cultures.

Eukaryotic chromosomes appear to consist of many replicons, the time of replication of which is probably controlled by specific origins. However, plasmids without specific eukaryotic origins may also replicate in some cells when injected into nuclei or transferred during transformation. The efficiency and the mechanisms of their initiation are still uncertain. A number of reports are cited which indicate that natural eukaryotic DNAs initiate their replication from specific origins. The nature of these origins are known in only a few instances and no general conclusions can yet be given about the nucleotide sequences involved. Short dispersed repeats of the Alu type appear to function as origins since they enhance the efficiency of replication of vector plasmids in Xenopus eggs. Certain sequences from a variety of eukaryotic DNAs also enhance the replicative potential of plasmids in yeast cells. The common features of such initiators or enhancers is uncertain. If dispersed repeats are origins in mammalian chromosomes, the number appears to be excessive. Either only a subset are functional, or the functional ones are only suborigins in larger replicons in which master origins (not yet isolated) function in the regulation of the timing of replication. Evidence is cited which indicates that the regulation of the time of replication of a gene or gene cluster is part of a regulatory system that makes the DNA available for transcription or leaves it in an inactive state. About one-half the DNA in mammalian cells is replicated in the first half of S phase (SE). After a brief pause in mid-S phase, the remainder of the DNA is replicated in what is designated late S (SL). The fractions replicated in SE and SL may vary in other phylogenetic groups, but wherever division of differentiated cells occurs such fractions are likely to be found. The following hypothesis is proposed. The DNA replicated in SL is suppressed in transcription, if it has the appropriate promoter regions, because the newly replicated DNA is complexed with proteins that suppress transcription. These proteins are only available during SL. Those genes replicated in SE are complexed with a different set of proteins which leave the promoter regions open for transcription when the appropriate regulatory molecules are available. In this way an inactive state or potentially active state can be transmitted from one cell generation to the next. Evidence is cited which indicates that genes which are active in all cells at some stage in the cell cycle are replicated in SE.(ABSTRACT TRUNCATED AT 400 WORDS)

A study of sedimentation and buoyant density of Okazaki fragments from mammalian chromosomes along with electron microscopic studies indicate that fragments from about 200 to 1200 nucleotides long may have RNA segments covalently attached. The fragments in some CsCl isopycnic gradients banded in two rather distinct bands. One band corresponds to the density of single-stranded DNA, but the other has a higher buoyant density which could be conferred by a segment of RNA up to 180 nucleotides or more in length. The RNA was not removed by denaturing conditions which separated DNA strands consisting of several thousand nucleotide pairs. When the material of higher buoyant density was spread for electron microscopy under conditions which would extend single-stranded DNA chains, but leave RNA in a coil or bush the chains with a higher buoyant density usually had a bush attached at one end. Under conditions that were thought to favor gap filling over chain elongation near growing forks, the DNA produced by pulse labeling with bromodeoxyuridine had a buoyant density which would indicate substitution to about 15 percent in one chain. If this substitution represents filling of gaps occupied by RNA before the pulse, the segments would be about 180 nucleotides in length assuming about 1,000 nucleotides between each segment.

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