Oligodeoxyribonucleotides complementary to the DNA of the wild type (wt) bacteriophage phi chi 174 have been synthesized by the phosphotriester method. The oligomers, 11, 14, and 17 bases long, are complementary to the region of the DNA which accounts for the am-3 point mutation. When hybridized to am-3 DNA, the oligonucleotides form duplexes with a single base pair mismatch. The thermal stability of the duplexes formed between wt and am-3 DNAs has been measured. The am-3 DNA:oligomer duplexes dissociate at a temperature about 10 degrees C lower than the corresponding wt DNA:oligomer duplexes. This dramatic decrease in thermal stability due to a single mismatch makes it possible to eliminate the formation of the mismatched duplexes by the appropriate choice of hybridization temperature. These results are discussed with respect to the use of oligonucleotides as probes for the isolation of specific cloned DNA sequences.

No abstract

No abstract

No abstract

The rat serum albumin gene has been isolated from a recombinant library containing the entire rat genome cloned in the X phage Charon 4A. Preliminary R-loop and restriction analysis has revealed that this gene is split into at least 14 fragments (exons) by 13 intervening sequences (introns), and that it occupies a minimum of 14.5 kilobases of genomic DNA.Recent advances in recombinant DNA technology have made it possible to obtain virtually any desired single-copy genomic sequence in cloned form, provided an appropriate probe is available. We have used these techniques to isolate the rat serum albumin gene. Serum albumin synthesis is one of the major characteristics of vertebrate liver. Observation of the activity and state of this gene during development and in adult tissues should be informative as to the process of terminal differentiation. Albumin synthesis is essentially constitutive, but does respond significantly to a variety of stimuli (1). It is also expressed to variable extents in different hepatoma cell lines (2). The availability of cloned albumin genomic DNA will greatly facilitate the study of this variable expression, particularly at the level of transcript processing.Determination of the sequence organization of the albumin gene is also of interest, especially with regard to the disposition of repetitive elements and intervening sequences. Although regulatory (3) and evolutionary (4) significance has been postulated, the functional role, if any, of these striking features of eukaryotic genomes remains unknown. The comparative studies that will be possible as other genes are extracted from the rat and related species can be expected to provide considerable insight into this fascinating problem. MATERIALS AND METHODSRat Genome Library. High molecular weight liver DNA was extracted from an adult male Sprague-Dawley rat (Simonsen Labs, Gilroy, CA) by the method of Blin and Stafford (5) and aliquots were digested with EcoRI (Boehringer Mannheim) under conditions adjusted to cleave either one-third or one-fifth of the EcoRI sites in an equivalent amount of bacteriophage X DNA. The fragments resulting from this partial digestion were sedimented through a 10-30% sucrose gradient; the material between 10 and 20 kilobases (kb) was recovered by ethanol precipitation. A sample of this rat DNA (2.5 ,g) was ligated with 8.5 ,ig of a preparation of Charon 4A "cloning fragments" (6, 7). This recombinant DNA was packaged in vitro by using extracts from defective X lysogens provided by N. Sternberg (6). The method used was that of Hohn and Murray (8). Approximately 2,000,000 independent clones were obtained. The library was amplified 100,000-fold by subconfluent plating on Escherichia coli strain DP5OSupF (9).cDNA Clones. cDNA was synthesized from purified albumin mRNA as described (10). This cDNA contained a small amount of full-length material and had a number average size of approximately 1000 nucleotides. It was rendered double-stranded by sequential treatment with E. coli DNA polymerase I and S1 nuc...

We have previously reportedl1 2 that chromatin isolated from pea embryos possesses the ability to carry out the DNA-dependent synthesis of RNA from the four riboside triphosphates.3 The present paper concerns the roles in such synthesis of the several components of chromatin. It will be shown that the DNA of pea embryo chromatin is present in at least two forms, namely, as DNA itself and as DNA bound in nucleohistone complex. It will be further shown that DNA fully complexed with histone is inactive in the support of DNA-dependent RNA synthesis.Materials and Methods.-Pea embryos: Pea seeds (var. Alaska) were germinated in 35-gallon barrels in lots of 25 lb. The seeds were soaked for 5 hr in running water at 200C and then gently sprayed with water for an additional 35 hr. The embryonic axes, approximately 1 cm in length, were next separated from the cotyledons in a semiautomatic 3-stage disassembly line. Fifty pound dry weight of seeds yield approximately 1 kg fresh weight of embryos.Preparation of chromatin: The chilled, sterilized (with 10OX diluted Clorox) embryos were ground for approximately 1 min in a Blendor with an equal weight of grinding medium (sucrose 0.25 M, tris pH 8.0,0.05 M, ,3-mercaptoethanol, 0.01 M, MgCl2, 0.001 M) and filtered successively through cheesecloth and miracloth to remove cell wall debris. The filtrate was then centrifuged for 30 min at 4,000 X g. Under these circumstances, mitochondria and smaller particles remain in suspension while starch and chromatin sediment. The gelatinous chromatin layer was scraped from the underlying, firm starch layer and washed by successive recentrifugation (10,000 X g) in grinding medium (1X), sucrose, 0.25 M (2X), and tris, 0.05 M, pH8.0(2X). ,3-Mercaptoethanol, 0.01 M, was included in all of the above media. The final pellet was resuspended in the tris buffer, 30 ml per kg initial embryos. The yield of such crude chromatin is approximately 0.5 gm per kg embryos; the yield of DNA, 50 mg per kg embryos.Purification of crude chromatin: The crude chromatin prepared as described above contains ca. 95% of the DNA of the embryo but is contaminated by nonchromosomal protein, removable by sucrose gradient centrifugation. This was accomplished by layering 5 ml of crude chromatin suspension on 25 ml of 2 M sucrose (0.01 M in j-mercaptoethanol). The upper third of the tube was then gently stirred to form a rough gradient and the tubes centrifuged in the SW-25 swinging bucket head at 20 krpm for 3 hr. The resulting pellet of which the major constituents are DNA and histone will be referred to as purified chromatin. Approximately 70% of the DNA of crude chromatin is recovered in the purified chromatin.Analyses: DNA and RNA were determined principally by the Schmidt-Tannhauser procedure according to Ts'o and Sato.4 The diphenylamine method of Burton6 was used for determination of DNA in the presence of much protein. Total protein was determined by the Folin-phenol method as described by Lowry.8 Histone protein was separated from total protein on the basis of the sol...

The isolated chromatin of higher organisms possesses several properties characteristic of the same chromatin in life. These include the presence of histone bound to DNA, the state of repression of the genetic material, and the ability to serve as template for the readout of the derepressed portion of the genome by RNA polymerase. The important respect in which isolated chromatin differs from the material in vivo, fragmentation of DNA into pieces shorter (5 x 10(6) to 20 x 10(6) molecular weight) than the original, does not appear to importantly alter such transcription. The study of isolated chromatin has already revealed the material basis of the restriction of template activity; it is the formation of a complex between histone and DNA. Chromatin isolated by the methods now available, together with the basis provided by our present knowledge of chromatin biochemistry and biophysics, should make possible and indeed assure rapid increase in our knowledge of chromosomal structure and of all aspects of the control of gene activity and hence of developmental processes.

No abstract