Genomic structure and expression of human beta-1,4-galactosyltransferase.

We have cloned 60 kilobases of overlapping human genomic DNA comprising the complete coding sequence of the biosynthetic enzyme beta-1,4-galactosyltransferase (GalTase). The human locus spans greater than 50 kb of genomic DNA and shows an exon structure similar to the mouse gene. However, contrary to the mouse and bovine systems, which yield two distinct transcripts, RNA blotting and RNase protection analysis of human mRNA derived from HeLa cells reveal only a single transcript, corresponding to the "short" form of the mouse and bovine transcripts.

Gonadotropin alpha subunit. Differential processing of free and combined forms in human trophoblast and transfected mouse cells.

The gonadotropins luteinizing hormone, follicle-stimulating hormone, and human chorionic gonadotropin are composed of two noncovalently linked subunits, alpha and beta. The alpha subunit, identical in all three hormones, is produced in excess over the unique beta subunits by pituitary and placenta, and is secreted as uncombined, or free subunit. Free alpha subunit from both tissues has a larger molecular weight than the dimer form.

Cloning and expression of a cDNA encoding a maize glutathione-S-transferase in E. coli.

The isolation and characterization of a family of maize glutathione-S-transferases (GST's) has been described previously. These enzymes are designated GSTs I, II and III based on size, substrate specificity and responsiveness to safeners. GST III has been shown to act on the herbicide alachlor as well as the commonly used substrate 1-chloro-2,4-dinitrobenzene (CDNB).

Structural analysis of a maize gene coding for glutathione-S-transferase involved in herbicide detoxification.

We have used the cDNA clone encoding maize glutathione-S-transferase (GST I) to isolate a genomic DNA clone containing the complete GST I gene. Nucleotide sequence analysis of the cDNA and genomic clones has yielded a complete amino acid sequence for maize GST I and provided the exon-intron map of its gene. The mRNA homologous sequences in the maize GST I gene consist of a 107 bp 5' untranslated region, a 642 bp coding region and ≈340 bp of the 3' untranslated region.

Messenger RNA encoding a glutathione-S-transferase responsible for herbicide tolerance in maize is induced in response to safener treatment.

Glutathione-S-transferases (GST's) in maize represent a family of enzymes which conjugate glutathione to several major classes of pre-emergent, selective herbicides. Chemicals termed safeners have been demonstrated to increase the tolerance of maize toward such herbicides when the maize seed has been previously treated with safeners. It has subsequently been shown that corresponding increases in glutathione-S-transferase species occur.

Purification of recA-based fusion proteins by immunoadsorbent chromatography. Characterization of a major antigenic determinant of Escherichia coli recA protein.

Monoclonal antibodies to Escherichia coli recA protein were prepared, characterized, and used as affinity reagents for the purification of recA and recA:somatostatin fusion proteins. The monoclonal antibodies recognize an antigenic determinant or determinants located between amino acids 260 and 330 of recA. Addition of a fragment of the recA gene coding for these amino acids to an unrelated gene (beta-galactosidase) allowed the resulting beta-galactosidase fusion protein to be recognized by the recA monoclonal antibodies.

Synthesis and glycosylation of the common alpha subunit of human glycoprotein hormones in mouse cells.

The synthesis and the post-translational modification of the alpha subunit of human glycoprotein hormones have been studied in a mouse cell. A full-length cDNA coding for the human alpha subunit has been expressed in mouse C127 cells under the control of mouse metallothionein regulatory sequences, using a bovine papilloma virus vector. Stable clones secreting the alpha subunit into the medium have been obtained.

Differential patterns in the temporal experession ofBombyx mori chorion genes.

cDNA clones for two distinct families ofBombyx mori chorion protiens, Class A (m2774) and Class Hc (m5000), were used to study the developmental regulation of specific chorion mRNAs. Individual follicles from maturing ovarioles were assayed by Northern blotting techniques and amino acid pulse-labeling to determine concomitant RNA accumulation and protein synthesis patterns with a temporal resolution of 2.2-2.

Purification and characterization of corn glutathione S-transferase.

Two glutathione S-transferase (GST) activities have been identified and purified from etiolated corn tissue. The first, designated GST I enzyme, is constitutively present in corn tissue, and the second, designated GST II enzyme, is present only in tissue which has been treated with chemical antidotes which protect corn against chloroacetanilide herbicides. The total activity constitutes approximately 2% of the soluble protein in these tissues.

The recombinant DNA technology.

The recombinant DNA technology or DNA cloning permits the isolation, amplification, and precise manipulation of specific DNA fragments. This is generally accomplished by linking or recombining the desired DNA fragment with a DNA molecule, termed the vector, which is capable of directing the replication of itself in a suitable host cell and any DNA segment covalently attached to it. Using this and associated technologies, it is possible to produce large amounts of specific proteins and to modify cell types by introducing the genes for proteins that are otherwise absent.

Hybridization-selected translation of Bombyx mori high-cysteine chorion proteins in Xenopus laevis oocytes.

Xenopus laevis oocytes were injected with poly(A)+-mRNA isolated from chorionating follicular epithelium of the domesticated silk moth (Bombyx mori). On two-dimensional gel electrophoresis, the resultant translation products comigrated with authentic, secreted, chorion standards, demonstrating that the frog oocyte system synthesizes and correctly process virtually all major chorion components. A cDNA clone has been shown to contain sequences complementary to those of mRNAs encoding B mori high-cysteine (Hc) chorion proteins Hc6-Hc11.

Multiple related immunoglobulin variable-region genes identified by cloning and sequence analysis.

We have identified at least six EcoRI fragments of mouse DNA that encode variable-region gene sequences closely related to the mouse kappa light chain, MOPC-149. Two of these fragments have been cloned, and the entire nucleotide sequence of the variable-region genes encoded on each has been determined. Both genes encode closely related variable-region sequences extending from codon position 1 through position 97.

A comparison of two cloned mouse beta-globin genes and their surrounding and intervening sequences.

The BALC/c mouse has two nonallelic beta-globin genes that appear to reside on two different Eco R1 fragments of genomic DNA. We have already cloned one of these fragments and shown that the gene encoded within it is interrupted by at least one large intervening sequence of DNA. We have now cloned and characterized the second beta-globin gene-containing fragment.

Use of an EK-2 vector for the cloning of DNA from higher organisms.

A certified EK2 bacteriophage lambda vector, which is useful for cloning fragments of DNA from higher organisms, is described.

The intervening sequence of a mouse beta-globin gene is transcribed within the 15S beta-globin mRNA precursor.

Mouse beta-globin in encoded in a discontinuous structural gene interrupted by a 550-base pair intervening sequence of DNA. Correspondingly, the mature beta-globin mRNA appears to be synthesized via a 15S precursor, the length of which roughly equals the total length of the coding and intervening sequences of the beta-globin gene. Using the electron microscope to visualize hybrid structures formed between this gene and the purified 15S beta-globin mRNA precursor, we show that the intervening sequence is present within the larger precursor molecule.

Intervening sequence of DNA identified in the structural portion of a mouse beta-globin gene.

The unusual electron microscopic appearance of a hybrid formed between 9S mouse beta-globin mRNA and its corresponding cloned gene segment is caused by at least one, and possibly two, intervening sequences of DNA that interrupt the mouse beta-globin gene. Such an interpretation is consistent with a paradoxical restriction site pattern previously noted in this gene and with the nucleotide sequence of that portion of the gene that spans both structural and intervening sequences. The large intervening sequence, approximately 550 base pairs in length, occurs in the structural globin sequence and immediately follows the beta-globin codon corresponding to amino acid 104.

Cloning specific segments of the mammalian genome: bacteriophage lambda containing mouse globin and surrounding gene sequences.

We have developed a general approach to the cloning of specific segments of the mammalian genome that involves a two-step purification of EcoRI fragments of mammalian DNA and their in vitro insertion into a suitably constructed EK2 derivative of bacteriophage lambda. The combination of fragment purification, exclusion of parental-type recombinants, and simple phage screening techniques permits the isolation of virtually any gene segment for which there is an identifying hybridization probe. We illustrate the approach by describing the cloning of an approximately 7000-base-long segment of mouse DNA containing globin and surrounding gene sequences.

In vitro packaging of a lambda Dam vector containing EcoRI DNA fragments of Escherichia coli and phage P1.

In this report we describe a coliphage lambda vector system for cloning endo R. EcoRI DNA fragments. This system differs significantly from those previously described in two ways.

EK2 derivatives of bacteriophage lambda useful in the cloning of DNA from higher organisms: the lambdagtWES system.

A derivative of bacteriophage lambda has been modified and tested together with an appropriate host system to meet the criteria of EK2 biologic containment for cloning DNA from higher organisms. In this report certain of the safety features are summarized and some of the tests carried out to confirm the containment properties of the vector are described. The cloning efficiency of this system, together with available gene purification and hybrid screening technology, indicate that it can be used to clone DNA fragments carrying specific, unique mammalian genes.

Purification and cloning of a mouse ribosomal gene fragment in coliphage lambda.

We have found and characterized a recombinant between the EK2 vector lambdagtWES.lambdaC and a portion of the mouse ribosomal genes. A 6.

Immunochemical evidence for glutamine-mediated degradation of glutamine synthetase in cultured Chinese hamster cells.

The specific activity of glutamine synthetase in cultured Chinese hamster cells is inversely related to the concentration of glutamine in the surrounding solution. Enzyme specific activity increases 8- to 10-fold when glutamine is removed from serum-free F12 growth media. The induction of glutamine synthetase activity occurs only after glutamine removal and not after the removal of other amino acids (methionine, leucine, or isoleucine).