Ned Mantei

The nucleotide sequence of a cloned human leukocyte Interferon cDNA

We have determined the nucleotide sequence of the human leukocyte interferon cDNA carried in hybrid plasmid Z-pBR322(Pst)/HcIF-2h, which has been shown to direct the formation of a polypeptide with human leukocyte interferon activity (Nagata et al., 1980). The 910 base pair insert contains a 567 (or 543) base pair coding sequence, which determines a putative preinterferon polypeptide consisting of a signal peptide of 23 (or less likely 15) amino acids, followed by an interferon polypeptide of 166 amino acids (calculated molecular weight, 19 390). The coding sequence is preceded by a (most likely incomplete) 56 bp leader and followed by a 242 bp trailer and seven A residues from the poly(A) tail: A comparison of the sequence of 35 amino terminal amino acids of lymphoblastoid interferon (Zoon et al., 1980; M. Hunkapiller and L. Hood, personal communication) and the corresponding sequence deducted for leukocyte interferon revealed 9 differences. This suggests that these two interferons are encoded by two non-allelic genes.

Sequence of the precursor of intestinal lactase-phlorizin hydrolase from fetal rat

The nucleotide sequence of the cDNA corresponding to the precursor of fetal rat intestinal lactase-phlorizin hydrolase was determined. The precursor consists of four tandemly organized homologous domains flanked by a signal peptide in the N-terminal region and by a transmembrane peptide in the C-terminal region.

EXPRESSION OF CLONED VIRAL AND CHROMOSOMAL PLASMID-LINKED DNA IN COGNATE HOST CELLS

Publisher Summary This chapter focuses on expression of cloned viral and chromosomal plasmid-linked DNA in cognate host cells. The possible hazards inherent to hybrid DNA technology are usually speculated. An important question in this regard is whether a viral genome linked to a prokaryotic vector can be expressed in a host cell. This is shown to be the case both for prokaryotic viral DNA in a prokaryotic host and for eukaryotic viral DNA in a eukaryotic host. It is found that an intact copy of Qβ DNA joined to pCRI elicits formation of Qβ in E. coli, a hybrid consisting of a head-to-tail polyoma DNA dimer and pBR322 causes polyoma formation in cultured mouse fibroblasts, and the cloned thymidine kinase (TK) gene of herpes simplex type I linked to pBR322 can transform TK− mouse L cells. The chapter discusses the properties of E. coli HB101 containing Qβ DNA plasmids. It also presents physical and biological characterization of a hybrid plasmid containing Qβ DNA and discusses the characterization of the Qβ RNA formed following infection with Qβ DNA-containing plasmids.

THE EXPRESSION OF A CLONED RABBIT CHROMOSOMAL β-GLOBIN GENE IN MOUSE L CELLS AND YEAST

Publisher Summary This chapter discusses an experiment to study the expression of a cloned rabbit chromosomal β globin gene in mouse L cells and yeast. In the experiment, the in vivo transcription of the extraneous rabbit β-globin gene was monitored by a modification of the Berk-Sharp procedure. From the experiment, it was estimated that the transformed L cell lines that were tested, in particular the one producing >2000 molecules rabbit β-globin mRNA (M2/R-l), contained less than 5O strands per cell of mouse β-globin messenger RNA. In the experiment, a specific probe was prepared and was hybridized to RNA (Ribonucleic Acid) sample. The RNA sample was analyzed by polyacrylamide gel electrophoresis. The probe discriminates between mouse and rabbit globin messenger RNA. To determine whether globin-specific RNA had been synthesized in the transformed yeast cells, total RNA was subjected to agarose gel electrophoresis under denaturing conditions. By comparing the intensity of the globin-specific RNA band with that of known amounts of α-globin mRNA, it was estimated that about 0.01% of the total yeast RNA is β-globin specific.

THE STRUCTURE OF CLONED RABBIT AND MOUSE β-GLOBIN GENES

Cloned β-globin genes of both mouse and rabbit each contain a large and a small intervening sequence (intron) of about equal length at precisely the same positions relative to the coding sequence. The homologous introns show some sequence similarity, particularly at the junctions with the coding sequence. They most likely arose from a common ancestral sequence and diverged substantially during evolution.