Motohiro Fuke

Characterization of cloned rat ribosomal DNA fragments

SummaryTwo Charon 4A lambda bacteriophage clones were characterized which contain all and part of the 18S ribosomal DNA of the rat. One clone contained two Eco RI fragments which include the whole 18S ribosomal RNA region and part of 28S ribosomal RNA region. The other clone contained an Eco RI fragment which covers part of 18S ribosomal RNA region. There were differences between the two clones in the non-transcribed spacer regions suggesting that there is heterogeneity in the non-transcribed spacer regions of rat ribosomal genes. The restriction map of the cloned mouse ribosomal DNA. Eco RI, Hind III, Pst I, and Bam HI sites in 18S ribosomal RNA region were in the same places in mouse and rat DNA but the restriction sites in the 5′-spacer regions were different.

HindIII-sensitive sites present once in every four repeats of EcoRI-sensitive sites in novikoff rat hepatoma DNA

Restriction endonuclease digestion followed by gel electrophoresis has been widely used to detect repeating sequences in DNA from many eukaryotes. Highly repeated fragments were found after restriction endonuclease treatment of DNA from several mammalian species, including calf, human, monkey, mouse, rat and sheep [l-4]. The a-component of African green-monkey DNA [S] was most extensively studied and the nucleotide sequence of the 172 basepair segment of the DNA cleaved by Hind111 restriction endonuclease has been determined [6]. The segment is repeated several million times in the genome. Multiples of a unit repeat of 376 base-pairs have been found [3] after HirzdIII restriction-endonuclease treatment of rat-liver DNA and small repeating fragments from 60-440 base-pairs found [l] after &oRI restrictionendonuclease treatment of rat-liver DNA. In the present study, DNA from Novikoff rat hepatoma cells was digested with 4 restriction endonucleases and analyzed by polyacrylamide gel electrophoresis. Interestingly, two repeating units, monomer and tetramer sizes, were found after treatment of the DNA with different restriction endonucleases. The fragments of the two repeating units come from the same GC-rich region of the DNA.

A T1 ribonuclease fragment present in 18 S ribosomal RNA of Novikoff rat ascites hepatoma cells and absent from 18 S ribosomal RNA of HeLa cells.

Oligonucleotides, produced by digestion of 32 P-labeled 18 S ribosomal RNAs from Novikoff rat ascites hepatoma and HeLa cells with T 1 ribonuclease, were mapped by homochromatography fingerprinting. The fingerprint patterns from the two rRNAs were quite similar and 18 of the separated large fragments had corresponding migrations but one fragment present in the rat 18 S rRNA was absent from the human 18 S rRNA. The nucleotide sequence assigned to this fragment is A-C-C-C-C-C-C-U-U-C-C-C-Gp.

Localization of highly repeated HindIII fragments of novikoff rat DNA to the nucleolus

Novlkoff rat ascltes hepatoma cells have large nucleoh compared to normal rat cells and nucleoli from these hepatoma cells have been isolated m high yield and purity [ 1 ] The nucleolus IS known to be the site of nbosomal RNA synthesis and the rlbosomal RNA genes are repeated lOO-SOO-tlmes/dlplold genome [ 1,2] It hds been estimated that the DNA content/nucleolus 1s 1 3 pg and that the DNA content/cell 1s 8 pg for Novlkoff rat cells [ 1 ] Rlbosomai DNA comprised 0 008% of the total DNA [3] and -0 05% of nucleoldr DNA The remaining 99 95% of nucleolar DNA has not yet been chdracterlzed Restriction endonuclease dIgestIon followed by gel electrophoresls has been used to detect repeated sequences m DNA from several mammahan species [4-61 Novlkoff rat hepatoma DNA was analyzed by gel electrophoresls after EcoRI restrIction endonuclease digestIon m this laboratory SrnallEcoRI fragments of Novlkoff rat DNA were exammed [7] by acrylamide gel electrophoresls and large EcuRI fragments of Novlkoff rat DNA [8] by agarose gel electrophorests The largest EcoRI fragment, fragment A, was reported [8] localized to the nucleolus Here, HlndIII digestIon patterns of total nuclear DNA, nucleolar DNA, and extranucleolar nucleoplasmlc DNA have been studied by agarose and acrylamlde gel electrophoresls From quantltatlve analysis of the amount of the smallest HzndIII DNA fragment m various fractions of the nucleus, this fragment has been found to be localized to the nucleolus 2 Materials and methods

Pseudogene IFN-αL: removal of the stop codon in the signal sequence permits expression of active human interferon

Biologically active interferon (10(6)-10(7) units/liter) was produced in Escherichia coli from modified human alpha interferon (IFN-alpha) pseudogene L. IFN-alpha pseudogene L has a stop codon in the signal peptide coding region. The region that contains the stop codon was replaced with the corresponding region of another human IFN-alpha gene, WA, that does not have a stop codon and was previously engineered for expression by fusion to the M13mp11 lac promoter. The interferon L fusion product was induced with IPTG after infecting E. coli JM103 with the M13 bacteriophage that contained the modified human IFN-alpha pseudogene L. Hence, the IFN-alpha L mature interferon coding sequence, which is not identical to any other alpha-interferon gene, has been conserved for active interferon coding information.

Partial methylation of 18 S ribosomal RNA detected by T1 ribonuclease digestion and homochromatography fingerprinting.

Studies on methylation of oligonucleotides of ribosomal RNA have shown quantitative [ 1,2] , species [l] and growth related variations [2]. In previous studies on the oligonucleotides produced by digestion of 32P-labeled 18 S rRNA from four mammalian species with Tl RNAase and mapped by homochromatography fingerprinting, j2P-content in the spots of the large oligonucleotides was determined and the molar yield was calculated [3,4]. The molar yield was approximately one for each large oligonucleotide except oligonucleotides 3 and 6 [4]. Oligonucleotide 3 was found in less than one molar yield in the 18 S rRNA of rat, mouse, hamster and man. Oligonucleotide 6 was found at approximately 10% molar yield in the four 18 S rRNAs. The nucleotide composition of oligonucleotide 3 was AaC6U4,GmA,UmC,G and of oligonucleotide 6 it was AJC6U4,UmC,G [4]. Both the ohgonucleotides contained the same 2’-@methylated dinucleotide, UmC, and A3C6U4G. Oligonucleotide 3 contained the extra 2’.O-methylated dinucleotide, GmA. The present study shows that a partial 2’.0methylation at the GmA position is responsible for the appearance of the two spots, and that this methylation is quantitatively similar in several species.

Nucleotide sequence of the 5′-terminal region of rat 18S ribosomal DNA

SummaryThe 5′-terminal 597 base-pairs (bp) of the Sprague-Dawley rat 18S ribosomal RNA gene and 10 bp of the adjoining transcribed spacer have been sequenced. Previously sequenced 10 large oligonucleotides of rat 18S RNA were located in this region. This mammalian sequence has been compared with the known sequences of yeast and frog 18S rDNAs. The analysis indicates that 534 bp of the 597 bp (89%) are conserved between rat and frog sequences but only 75% of the nucleotides are conserved between rat and yeast in this region. Two large and two small sections have been identified where insertions have been introduced during evolution. Of these 58 bp long inserted sections of the rat rDNA sequence, 50 bp (86%) were G-C base-pairs.

[53] A procedure for isolation of alpha interferon genes with short oligonucleotide probes

Publisher Summary The chapter describes a procedure for the isolation of alpha interferon genes with short oligonucleotide probes. A method has been developed, which permitted the isolation of several human alpha interferon (IFN-α) genes from a human genomic library in Charon 4A bacteriophages with 17-base synthetic oligonucleotide probes. Previously, human IFN-α genes are isolated from complementary DNA (cDNA) libraries or genomic libraries with cDNA probes. The use of select, short, synthetic probes permits the isolation of the subsets of genes within a gene family represented in a genomic library. The screening procedure with 17-base probes described in the chapter is useful for the isolation of most genes from a human genomic library, although some conditions will vary according to the sequence of the probes. Procedure yielded three IFN-α genes, which had perfect homology with the two probes utilized. Even genes containing introns could be isolated from a genomic library with short and mixed probes provided the intron does not split the sequences of the probes to a size unable to maintain a stable hybrid. Changes at positions 42 and 133 result in the replacement of acidic residues with hydrophobic residues.

A rapid procedure to determine the content of 2′-O-methylation in RNA by homochromatography

Abstract The content of 2′- O -methylated dinucleotides from 17 S rRNA of yeast and 18 S rRNAs of chicken cells and Novikoff rat cells was determined by a new procedure using homochromatography. The procedure is simple and can be used to compare the extent of 2′- O -methylation simultaneously in various RNAs. It was of interest that 18 S rRNAs of chicken and rat have two times more 2′- O -methylated dinucleotides as compared to yeast 17 S rRNA.