Nucleotide sequence of the simian virus 40 Hind II + III restriction fragment J and the total amino acid sequence of the major structural protein VP1.

The HindII + III restriction fragment J (Hind-J) represents 4.58% of the simian virus 40 genome. The information present in Hind-J is expressed as part of the major, late 16-S messenger RNA, which codes for the structural protein VP1. The nucleotide sequence of the 240-base-pairs-long Hind fragment J has been determined by analysis of each oligonucleotide from both strands resulting from T1 or pancreatic RNase digestion of RNA transcribed from the DNA and from RNase digestion of ribo-substituted DNA. Large oligonucleotide blocks which could be constructed mainly on the basis of complementarity were subsequently ordered by partial chemical degradation of terminally labeled DNA. This direct DNA sequencing approach also completely confirmed the results obtained by both aforementioned RNase degradation methods. In the strand with the same polarity as the late mRNA, triplets corresponding to termination codons are present in two of the three reading frames. The one open reading frame connects in phase with the open reading frame of the neighboring HindII/ III fragments K, F and G, which have been published previously and which together with Hind-J span the total VP1 gene. Some features of the primary nucleotide sequence of this VP1 gene and the derived VP1 amino acid sequence are discussed.     

Nucleotide sequence of the SV40 DNA restriction fragment Hind C-Hap 2.

We here report the nucleotide sequence of the SV40 DNA fragment Hind C - Hap 2. The fragment was labeled at the 5'-ends by means of polynucleotide kinase and gamma-(32P) ATP and digested with a suitable restriction enzyme. The separated products were then partially degraded with the base-specific reagents dimethyl-sulphate or hydrazine followed by direct analysis on polyacrylamide gel. The Hind C - Hap 2 sequence is 126 base pairs long and one of the three possible reading frames for translation does not contain any termination codon. So, although no protein is known to be encoded by this region, the possibility cannot yet be completely ruled out. The sequence also contains several AT-rich blocks.     

DNA sequence variants in the G gamma-, A gamma-, delta- and beta-globin genes of man

DNA prepared from 60 unrelated individuals was cleaved with one of eight different restriction endonucleases and the resulting DNA fragments were separated by agarose gel electrophoresis. DNA fragments containing G gamma-, A gamma-, delta- or beta-globin genes were detected by Southern blot hybridization, using as probe either a 32P-labeled cloned DNA copy of rabbit beta-globin messenger RNA or labeled human beta- and G gamma- globin cDNA plasmids. Three types of variant restriction enzyme patterns of globin DNA fragments were detected in otherwise normal individuals. One variant pattern, found in only one person, was caused by an additional restriction endonuclease Pst I cleavage site in the center of the delta- globin gene intervening sequence; the subject was heterozygous for the presence of this cleavage site and was shown to have inherited it from her mother. Another variant pattern resulted from the appearance of an endonuclease Hind III cleavage site in the intervening sequence of the A gamma-globin gene; this variant is polymorphic, with a gene frequency for the presence of the intragenic Hind III site of 0.23. This Hind III cleavage site polymorphism is also found in the G gamma-globin gene intervening sequence and thus the polymorphism itself appears to be duplicated over the pair of gamma-globin loci. These variants can be used to derive an approximate estimate of the total number of different DNA sequence variants in man.     

Determination of the sequences of 18 nucleotides from the 5''-end of the 1-strand and 15 nucleotides from the 5''-end of the r-strand of T7 DNA.

The sequences of 18 nucleotides from the 5'-end of the 1-strand and 15 nucleotides from the 5'-end of the r-strand of T7 bacteriophage DNA have been determined to be pT-C-T-C-A-C-A-G-T-G-T-A-C-G-T-C-C-C (1-strand) and pA-G-G-G-A-C-A-C-A-G-C-G-C-T-C (r-strand). The 5'-termini of whole DNA or separated strands were kinased using polynucleotide kinase and (gamma-32-P) rATP. The DNA was partially digested with pancreatic DNase and the fragments were separated by two dimensional electrophoresis and homochromatography. To complete the sequence, snake venom phosphodiesterase digestions of these fragments were carried out. The relationship of these sequences to the proposed cleavage of concatemeric DNA during DNA replication is discussed.     

Rifampicin-resistant initiation of DNA synthesis on the isolated strands of ColE plasmid DNA.

The opposite strands of the ColE1 and ColE3 plasmids were isolated as circular single-stranded DNA molecules. These molecules were compared with M13 and phi X174 viral DNA with respect to their capacity to function as templates for in vitro DNA synthesis by a replication enzyme fraction from Escherichia coli. It was found for both ColE plasmids that the conversion of H as well as L strands to duplex DNA molecules closely resembles phi X174 complementary strand synthesis and occurs by a rifampicin-resistant priming mechanism involving the dnaB, dnaC, and dnaG gene products. Restriction analysis of partially double-stranded intermediates indicates that preferred start sites for DNA synthesis are present on both strands of the ColE1 HaeII-C fragment. Inspection of the nucleotide sequence of this region reveals structural similarities with the origin of phi X174 complementary strand synthesis. We propose that the rifampicin-resistant initiation site (rri) in the ColE1 L strand is required for the priming of discontinuous lagging strand synthesis during vegetative replication and that the rri site in the H strand is involved in the initiation of L strand synthesis during conjugative transfer.     

Identification and isolation of transcribed human X chromosome DNA sequences

A human X chromosome specific phage library has been used as a source of X-specific genomic DNA clones which hybridize with cellular RNA. Random cDNA clones were mapped for X chromosome sequence localization and 8 were identified as hybridizing to X chromosome Hind III fragments. All eight also hybridized with autosomal Hind III fragments. The X chromosome genomic sequences corresponding to two of these cDNA clones were isolated from a phage library constructed with the Hind III endonuclease digest products of X enriched DNA. One genomic DNA segment, localized to the short area of the X, shared sequence homology with at least one region of the human Y chromosome. The methodology developed represents a rapid means to obtain a specific genomic DNA clone from a single chromosome when multiple different genomic loci homologous to an expressed DNA sequence exist.     

Separation of complementary strands of plasmid DNA using the biotin-avidin system and its application to heteroduplex formation and RNA/DNA hybridizations in electron microscopy.

A method for the separation of complementary strands with the help of the biotin-avidin system is described. Restriction fragments were terminally labeled at both ends with biotinylated nucleotides. The DNA was cut by a second restriction enzyme, and the fragments were bound to an avidin agarose column. The non-biotinylated strands were eluted with 0.1 M NaOH, and the biotin-labeled strands were subsequently released from the column by elution with 50% guanidine isothiocyanate/formamide. Contamination of the separated strands by complementary single strands was less than 4%.-Separated linear single strands of the vector pEMBL were prepared. On annealing with recombinant circular DNA a substitution loop is formed which provides position and orientation markers for the unambiguous electron microscopic analysis of heteroduplexes or hybrids formed with the inserted sequences. -The terminal biotin label was visualized by complex formation with a streptavidin-ferritin conjugate.     

Restriction fragment length polymorphism of the rat albumin gene in Sprague-Dawley rats and its application in genetic study of analbuminemia.

Restriction fragment length polymorphism of the rat albumin gene was discovered in a stock of Sprague-Dawley rats by Southern blots of rat liver DNAs using cloned albumin cDNA, prAlb-1 (1), as a probe. The polymorphic DNA fragments were observed when rat DNAs were digested with either Hind III or Pst 1 and the difference in length of the DNA fragments in Hind III or Pst 1 digests was estimated as 1.4 kbp. When DNAs were digested with EcoR I, restriction fragment length polymorphism was not observed. Therefore, this polymorphic DNA was concluded to be located in the flanking sequence. Structural analysis of the cloned albumin gene showed that the polymorphism was located in the 3'-flanking sequence. With this polymorphism as a marker of the albumin structural gene, the phenotype of analbuminemia, which is an autosomally recessive trait, was found to be linked to the structural gene of albumin.     

Molecular characterization of Penicillium expansum MUCL mutants

The modern biomolecular analysis of DNA was carried out to determine the identity of Penicillium expansum MUCL V1-V9 Variants with parental strain of Penicillium expansum MUCL 29412 and to compare the results with Penicillium verrucosum, a related species. The extracted DNAs were fragmented by digestion with restriction endonuclease Hind III and the fragments were separated by agarose gel electrophoresis. The DNAs were then denaturated in the gel after partial depurination with dilute acid and were transferred to a nylon membrane. The membrane was incubated with 32P-labeled probe, which was a DNA having a base sequence complementary to the DNA that was to be detected on the filter. The hybridization of the restriction fragments was performed for a highly qualitative comparison of the digested fragments. The analysis of the DNA profile, the most important stage in DNA identity testing, confirms the identity of the DNA for all strains of Penicillium expansum MUCL, except for V5 Variant.     

Replication origin of the Bacillus subtilis chromosome determined by hybridization of the first-replicating DNA with cloned fragments from the replication origin region of the chromosome

The replication origin (ori) on the Bacillus subtilis genome was determined by the hybridization between the first-replicating DNA region and the cloned fragments from the ori region. The first-replicating DNA region was labeled specifically by [³H]thymidine in the presence of an inhibitor for DNA polymerase during a synchronous initiation of the chromosomal replication by germinating spores starved for thymine, and isolated by a sucrose density gradient centrifugation. Most of the labeled DNA molecules are small in size (up to 1000 bases long). The 45-kb ori region was cloned first in a λ Charon vector and then subcloned in pBR vectors. Restriction fragments from these cloned DNAs were purified by electrophoresis in agarose gels.

Only one region within the 45-kb ori region shows strong hybridization with the first-replicating DNA. Restriction fragments from this region were cloned in a phage M13 vector and separated into complementary strands. Hybridization of the labeled DNA with these cloned single-stranded fragments revealed that one site of the ori is located in each strand and they are some 2-kb apart from each other. Replication starts from these sites and proceeds inwards to pass each other.     

Nucleotide sequence definition of a major human repeated DNA, the Hind III 1.9 kb family

A human Hind III 1.9 kb repeated DNA fragment was isolated and cloned in pBR322. A cloned member that hybridized predominantly to the 1.9 kb Hind III band in a digest of whole human DNA was chosen for sequencing. It is an 1894 bp fragment that shows no significant internal repeats. Few pCG residues are observed in the sequence and there are numerous stop codons. Detailed sequence comparisons confirm this is a novel class of repeats that is not related to previously characterized human satellite DNAs or Alu sequences. At least a portion of the sequence described is conserved in evolution.     

Coding capacity of complementary DNA strands.

A Fortran computer algorithm has been used to analyze the nucleotide sequence of several structural genes. The analysis performed on both coding and complementary DNA strands shows that whereas open reading frames shorter than 100 codons are randomly distributed on both DNA strands, open reading frames longer than 100 codons ("virtual genes") are significantly more frequent on the complementary DNA strand than on the coding one. These "virtual genes" were further investigated by looking at intron sequences, splicing points, signal sequences and by analyzing gene mutations. On the basis of this analysis coding and complementary DNA strands of several eukaryotic structural genes cannot be distinguished. In particular we suggest that the complementary DNA strand of the human epsilon-globin gene might indeed code for a protein.     

Sequence analysis of the major outer membrane protein gene from Chlamydia trachomatis serovar L2.

The structural gene for the major outer membrane protein (MOMP) from Chlamydia trachomatis was cloned and sequenced. A lambda gt11 recombinant (lambda gt11/L2/33) that contains a portion of the MOMP coding sequence was used to probe a lambda 1059 library constructed from DNA obtained from C. trachomatis serovar L2. Selected lambda 1059 recombinants were mapped with endonuclease restriction enzymes. The MOMP gene was mapped to the 5' end of a BamHI fragment of approximately 9 kilobases. Contiguous endonuclease restriction fragments identified within this region permitted the selection of specific fragments for subcloning and DNA sequencing. The MOMP gene consisted of a 1,182-base-pair open reading frame that encoded 394 amino acids and ended with three stop codons. The known amino-terminal amino acid was preceded by 22 amino acids whose sequence was compatible with a leader or signal sequence. The primary structure of MOMP determined from the translated DNA sequence demonstrated nine cysteine residues and a remarkably homogeneous distribution of charged and hydrophobic residues.     

A complete cleavage map of Neurospora crassa mtDNA obtained with endonucleases Eco RI and Bam HI.

A physical map of Neurospora crassa mitochondrial DNA has been constructed using specific fragments obtained with restriction endonucleases. The DNA has 5 cleavage sites for endonuclease Bam HI, 12 for endonuclease Eco RI and more than 30 for endonuclease Hind III. The sequence of the Eco RI and Bam HI fragments has been established by analysis of partial fragments. By digestion of the Eco RI fragments with Bam HI, a complete overlapping map has been constructed. The position of the largest Hind III fragment on this map has also been determined. The map is circular and the added molecular weight of the fragments is 40 - 10(6), which is in good agreement with earlier measurements on intact DNA, using the electron microscope.     

Nucleotide sequence of simian virus 40 DNA: structure of the middle segment of the HindII + III restriction fragment B (sixth part of the T antigen gene) and codon usage

We report here the nucleotide sequence of the simian virus 40 DNA region that lies between the EcoRII restriction endonuclease cleavage sites at map positions 0.214 and 0.281. The sequence was determined by partial chemical degradation of terminally labeled DNA fragments according to the procedure of Maxam and Gilbert. This region represents 6.7% of the SV40 genome and is located in the middle of HindII + III restriction fragment B. It is expressed as part of the early 19-S messenger RNA, which codes for the large-T antigen protein. Only one open reading frame for translation can be deduced from the message strand of the DNA and this reading frame connects in phase with the one of both neighboring fragments. This publication is the last in a series of papers about the T-antigen gene, and several properties of this gene and its product are discussed. The non-randomness of codon usage is similar to that previously discussed for the late part of the genome. Moreover, it appears that the choice of a third letter can be determined by the nature of the following codon; some codons which start with a pyrimidine are almost never preceded by an adenosine and some ANN-type codons are almost never preceded by a guanosine.