Cloning of alkaline phosphatase isozyme gene (iap) of Escherichia coli

In Escherichia coli three major alkaline phosphatase isozymes are formed by molecular conversions depending on physiological conditions. A chromosomal gene, iap, is responsible for alkaline phosphatase isozyme conversion and is assumed to code for a proteolytic enzyme removing the arginine residue(s) from the N-terminal position of alkaline phosphatase subunits. A chromosomal fragment which complemented the Iap^? phenotype was cloned into pBR322 by a shotgun method. Transducing phage λiap was constructed in vitro from the chromosomal fragment containing the iap gene and λtna DNA. The integration site of the phage on chromosome was identified as the iap locus by PI transduction, which meant that the cloned chromosomal DNA contained authentic iap gene.The restriction map of the hybrid plasmid was constructed. Based upon this information, several iap deletion plasmids as well as smaller iup⁺ plasmids were constructed. Analysis of the phenotypes conferred by these plasmids enabled us to locate iap gene within a 2-kb segment of the cloned DNA.The cells carrying the iap⁺ plasmid showed very efficient isozyme conversion even in medium containing arginine, an inhibitor for the isozyme conversion. This indicates overproduction of the iap gene product.     

Construction of recombinant plasmids containing rat thyroglobulin mRNA sequences

Two plasmids containing rat thyroglobulin cDNA sequences have been constructed and characterized. A plasmid with a 500-bp insert (pRT6) was isolated and identified as thyroglobulin-specific on the basis of the tissue specificity of the inserted sequence and of its ability to retain thyroglobulin mRNA on a nitrocellulose filter. The cDNA insert in pRT6 was subsequently used to screen a rat thyroid cDNA library constructed with large cDNA. A plasmid was found containing a 1700-bp insert. The polarity and the fidelity of the insert is demonstrated by S1 mapping.     

Cloning of the ColE3-CA38 colicin and immunity genes and identification of a plasmid region which enhances colicin production

The colicin and immunity genes of plasmid ColE3-CA38 have been localized by characterization of bacteria carrying its cloned restriction fragments. They are within a 3.14-kb EcoRI segment, such that the immunity gene contains the KpnI site, and the colicin gene is adjacent to it within a 2.1-kb KpnI-HincII segment. The immunity gene and one end of the colicin gene are in the region of ColE3-CA38 which is not homologous to the closely related plasmid ColE2-P9. A 0.64-kb PvuI-EcoRI segment of the plasmid adjacent to that containing the colicin and immunity genes was found to augment colicin production on solid media, and also affected the morphology of clearing zones produced by the cells when used as indicators in overlays of stabs of colicin E2 or E7 producers. The 0.64-kb segment was required in its native orientation relative to the 3.14-kb EcoRI segment to cause its effects.     

Identification of N- and C-terminal corticotropin peptides in the Mr 80000 form of neurophysin

The 125I-labeled Mr 80000 form of neurophysin has been purified from bovine neurohypophysi. Tryptic digests of this species were analyzed, prior to or after treatment with carboxypeptidase B, by high-pressure liquid chromatography followed by isoelectric focusing and the fragments compared with those generated by a similar treatment of reference bovine 1-39 adrenocorticotropin. The ACTH peptides 22-39 and 1-8, as well as the 1-7 derivative of the latter were identified by those two independent criteria. This provides chemical evidence supporting the hypothesis [8] that high Mr neurophysin may contain the sequence of ACTH.     

Phytochrome control of plastid mRNA in mustard (Sinapis alba L.)

The steady-state levels of plastid RNA sequences in dark-grown and light-grown mustard (Sinapis alba L.) seedlings have been compared. Total cellular RNAs were labeled in vitro with ³²P and hybridized to separated restriction fragments of plastid DNA. Cloned DNA fragments which encode the large subunit (LS) of ribulose-1,5-bisphosphate carboxylase [3-phospho-D-glycerate carboxylase (dimerizing), EC 4.1.1.39] and a 35,000 plastid polypeptide were used as probes to assess the levels of these two plastid mRNAs. The 1.22-kilobase-pair mRNA for the 35,000 polypeptide is almost undetectable in dark-grown seedlings, but is a major plastid mRNA in light-grown seedlings. The hybridization analysis of RNA from seedlings which were irradiated with red and far-red light indicates that the level of this mRNA, but not of LS mRNA, is controlled by phytochrome.Abbreviations LS large subunit - RuBP ribulose-1,5-bisphosphate - ptDNA plastid DNA     

Genetic mapping of bovine mitochondrial DNA from a single animal

Using a physical map of bovine mitochondrial DNA derived from the liver of a single Holstein cow, we have determined the location of the genes specifying the large and small riibosomal RNAs by hybridization analysis and electron microscopic observations of R-loop forms. Also, the position of the origin of DNA replication (D-loop) has been located by electron microscopy. Additionally, the direction of D-loop expansion and the polarity of the large and small ribosomal RNA genes were determined.     

A sequence specific endonuclease from Micrococcus radiodurans

A new sequence specific endonuclease, MraI has been purified from Micrococcus radiodurans. This enzyme cleaves bacteriophage λ DNA at three sites, adenovirus type 2 DNA at more than 12 sites and has a unique site on ΦX174 DNA. It has no sites on SV40, PM2 and pBR322 DNA. The three sites on phage λ DNA are different from those cleaved by SmaI, XmaI and XorII. The sites of cleavage are located at 0.424, 0.447 and 0.834 fractional lengths on the physical map of λ DNA. MraI is shown to be an isoschizomer of SacII and SstII recognizing the palindromic nucleotide sequence ′5-CCGC↓GG-3′. The enzyme shows an absolute requirement of Mg²⁺, but is active in the absence of added 2-mercaptoethanol. The enzyme shows activity at a broad range of temperature and pH with an optimum at 45°C and pH 7.0. MraI represents the first restriction enzyme from a bacterium whose DNA lacks modified methylated bases.     

Assignment of the sorbitol dehydrogenase locus to human chromosome 15 pter→q21

The locus for sorbitol dehydrogenase (SORD, E.C. 1.1.1.14) has been shown to segregate with hexosaminidase A and mannose phosphate isomerase in a series of human-Chinese hamster somatic cell hybrids. Cytogenetic analysis supports the assignment to chromosome 15 and further defines the gene locus to the region 15pterq21.This research was supported by the Medical Research Council of Canada (MT 4061), the Children's Hospital of Winnipeg Research Foundation, Inc., and the Department of Health, Province of Manitoba (H.S.W.).     

Genetics of formamidase-5 (brain formamidase) in the mouse: Localization of the structural gene on chromosome 14

A single formamidase, which is different from the formamidases found in other tissues, occurs in the brains of mice. This enzyme is here called formamidase-5 and the gene symbol is designated For-5. Two alleles are recognized on the basis of their differential heat sensitivity: For-5 ^b is relatively heat stable and is present in strain C57BL/6J, while For-5 ^d is relatively heat sensitive and is present in strain DBA/2J. The heat sensitivity of formamidase-5 in 44 other inbred strains and substrains was tested and found to resemble that of C57BL/6J or DBA/2J. Thirty-six recombinant inbred strains derived from progenitors that differed at For-5 were studied to test for single-gene inheritance and linkage with other loci. Complete concordance was found with the esterase-10 locus (Es-10), indicating close linkage. The 99% upper confidence limit of the distance between For-5 and Es-10 is 3.7 centimorgans (cM). Es-10 is located on chromosome 14 about 19 cM from the centromere. An independent demonstration of linkage of For-5 with Es-10 and another chromosome 14 marker, hairless (hr), is provided by the finding that the HRS/J strain, which has been sibmated for 60 generations with forced heterozygosity at the hr locus, is cosegregating at For-5 and Es-10. A survey of 32 inbred strains and substrains revealed that the For-5 ^d allele is associated with the Es-10 ^b allele, and that the For-5 ^b allele is associated with Es-10 ^a and Es-10 ^c. Formamidase-5 segregates as expected in the F₂ generation of crosses between strains bearing For-5 ^b and For-5 ^d alleles. It is possible that this unique formamidase of the brain is involved in the metabolism of a neurotransmitter substance.This research was sponsored in part by the Department of Energy under contract with the Union Carbide Corporation and in part by NIH Research Grant GM-18684 from the National Institute of General Medical Sciences. J. C. F. is a predoctoral Fellow supported by Grant CA 09104 from the National Cancer Institute. The Biology Division of Oak Ridge National Laboratory and the Jackson Laboratory are fully accredited by the American Association for Accreditation of Laboratory Animal Care.