Cloning of cDNA for human T-cell replacing factor (interleukin-5) and comparison with the murine homologue.

We have cloned cDNA for T-cell replacing factor (interleukin-5), which replaces T-cell helper function for normal B cells which secrete immunoglobulin, from human T cell leukemia line, ATL-2, using mouse interleukin-5 cDNA as probe. Total nucleotide sequence of the cDNA (816 base pairs) was determined and compared with that of mouse interleukin-5 cDNA. The cloned cDNA encoded the interleukin-5 precursor of 134 amino acids containing an N-terminal signal sequence. Although the human interleukin-5 precursor is one amino acid longer than the murine homologue, the sizes of the mature proteins appear similar. The nucleotide and amino acid sequence homologies of the coding regions of human and murine interleukin-5 are 77% and 70%, respectively. Human interleukin-5 synthesized by the direction of the cloned cDNA induced immunoglobulin synthesis in human B cells stimulated by Staphylococcus aureus mitogen.     

Cloning and structure of a mouse interleukin-2 chromosomal gene

Using non-stringent hybridization with a human interleukin-2 cDNA probe, we have isolated recombinant phages from a mouse genomic DNA library cloned in the EMBL3 phage. The sequence and organization of the mouse interleukin-2 (IL-2) gene was determined. By comparison with the human IL-2 sequence, three introns can be identified with lengths of 99, ±2 400, and ±1 900 base pairs, respectively. The mouse IL-2 gene codes for a polypeptide of 169 amino acids and contains a putative signal peptide of 20 amino acids. The homology to the human interleukin-2 is 72% at the nucleotide level in the coding part and 65% at the amino acid level. An extraordinary sequence, consisting of 12 consective CAG codons coding for glutamine, is found in the first exon.     

The human transferrin gene: 5' region contains conserved sequences which match the control elements regulated by heavy metals, glucocorticoids and acute phase reaction

Transferrin is a major plasma protein that transports iron to proliferating cells throughout the body. A clone containing the 5' region of the human transferrin gene has been isolated and characterized. A 14 kb EcoRI fragment was identified that contained the first 8 exons of the transferrin gene and 3.6 kb of its 5' flanking region. Conserved sequences identical or homologous to regulatory elements responding to heavy metals, glucocorticoid receptor and a putative acute phase reaction signal were identified in the 5'flanking region and intron 1. Also, the regulatory region of the transferrin gene contains a 14-bp sequence which closely matches sequences found in the interleukin-2 and gamma-interferon genes. All three genes are expressed by T lymphocytes before proliferation. A secondary loop structure similar to that proposed for the ovotransferrin gene can be formed by sequences in the 5' untranslated region of the transferrin mRNA.     

Secretion of human EGF and IgE in mammalian cells by recombinant DNA techniques; use of a IL-2 leader sequence

Expression plasmids were constructed containing chemically synthesized human epidermal growth factor (EGF) gene fused in a frame to a leader sequence of human interleukin-2 (IL-2) gene under the control of a viral promoter. COS7 cells transfected with the plasmids synthesized and secreted EGF. Transfection of mouse A9 cells or BALB/3T3 clone A31 cells with the plasmids permitted the isolation of cell lines secreting the product which showed EGF activity. In particular, A31 transformed cells secreting human EGF grew well even in a medium containing a minimal level of serum. Using similar vectors having IgE cDNA (C2-C4) in place of EGF gene, a human IgE Fc fragment was also produced and secreted in mouse cells. These results show that heterologous leader sequences are useful for the expression and secretion of proteins whose genes lack leader sequences.     

Chemical synthesis of a gene for human stefin A and its expression in E. coli

A DNA containing the coding sequence for the human cysteine proteinase inhibitor stefin A was obtained by enzymic ligation of chemically synthesized deoxyoligonucleotides, using the Khorana ligation method. The 306-bp synthetic gene carries signals for the initiation and termination of its translation. The gene was expressed in E. coli using a cytoplasmic expression vector and stefin A was secreted under the control of the E. coli alkaline phosphatase signal sequence, respectively. The secreted hybrid protein was shown to exhibit biological properties similar to the native protein isolated from human plasma.     

Molecular cloning of human interleukin 2 cDNA and its expression in E. coli.

A recombinant plasmid containing human interleukin 2 (IL2) cDNA was identified in a cDNA library constructed from mRNA derived from PHA-TPA induced splenocytes. Using this cDNA as a hybridization probe, a DNA fragment containing the IL2 gene was isolated from a collection of hybrid phages derived from human genomic DNA. A unique reading frame was identified from the nucleotide sequence derived from these plasmids coding for a polypeptide of 153 amino acids and containing a putative signal sequence of 20 amino acids. A mature polypeptide starting with either Met-Ala-Pro or Met-Pro was expressed in E. coli under control of the E. coli trp promoter or using a combination of the phage lambda PL promoter and a ribosome binding site derived from phage Mu. The bacterial IL2 polypeptide had a molecular weight of 15,000 daltons and accounted for more than 10% of the total E. coli proteins in fully induced cells; it was biologically active in the T-cell specific DNA synthesis assay, even after recovery from a SDS-containing polyacrylamide gel.     

Saccharomyces cerevisiae secretes and correctly processes human interferon hybrid proteins containing yeast invertase signal peptides.

Synthetic oligonucleotides coding for the yeast invertase secretion signal peptide were fused to the gene for the mature form of human interferon (huIFN-alpha 2). Two plasmids (E3 and F2) were constructed. E3 contained the invertase signal codons in a reading frame with the mature huIFN-alpha 2 gene. F2 had a deletion of the codon for alanine at amino acid residue-5 in the invertase signal and an addition of a methionine codon located between the coding sequences for the invertase signal and mature huIFN-alpha 2. Both hybrid genes were located adjacent to the promoter from the 3-phosphoglycerate kinase gene on the multicopy yeast expression plasmid, YEp1PT. Yeast transformants containing these plasmids produced somewhat more IFN than did the same expression plasmid containing the IFN gene with its human secretion signal sequence. HuIFN-alpha 2, purified from the medium of yeast cells containing E3, was found to be processed at the correct site. The huIFN-alpha 2 made by plasmid F2 was found to be completely processed at the junction between the invertase signal (a variant) and the methionine of methionine-huIFN-alpha 2. These results strongly suggested that the invertase signal (or its variant) attached to huIFN was efficiently recognized by the presumed signal recognition particle and was cleaved by the signal peptidase in the yeast cells. These results also suggested that amino acid changes on the right side of the cleavage site did not necessarily prevent cleavage or secretion.     

Human spermidine synthase gene: structure and chromosomal localization

The human spermidine synthase (EC 2.5.1.16) gene was isolated from a genomic library constructed with DNA obtained from a human immunoglobulin G (IgG) myeloma cell line. Subsequent sequence analyses revealed that the gene comprised of 5,818 nucleotides from the cap site to the last A of the putative polyadenylation signal with 8 exons and 7 intervening sequences. The 5'-flanking region of the gene was extremely GC rich, lacking any TATA box but containing CCAAT consensus sequences. No perfect consensus sequence for the cAMP-responsive element for the AP-1 binding site was found, yet the gene contained seven AP-2 binding site consensus sequences. The putative polyadenylation signal was an unusual AATACA instead of AATAAA. Polymerase chain reaction analysis with DNA obtained from human x hamster somatic cell hybrids indicated that human spermidine synthase genomic sequences segregate with human chromosome 1. Transfection of the genomic clone into Chinese hamster ovary cells displaying a low endogenous spermidine synthase activity revealed that the gene was transiently expressed and hence in all likelihood represents a functional gene.     

Improved assay sensitivity of an engineered secreted Renilla luciferase.

We have previously reported the construction of a functional Renilla luciferase enzyme secreted by mammalian cells when fused to the signal peptide of human interleukin-2. The presence of three predicted cysteine residues in the amino acid sequence of Renilla luciferase suggested that its secreted form could contain oxidized sulfhydryls, which might impair enzyme activity. In this work, four secreted Renilla luciferase mutants were constructed to investigate this possibility: three luciferase mutants in which a different cysteine residue was replaced by an alanine residue, and one luciferase mutant in which all three cysteine residues were replaced by alanine residues. Simian cells were transfected with the genes encoding these mutant luciferases, as well as with the original gene construct, and cell culture media were assayed for bioluminescence activity. Only media containing a mutated luciferase with a cysteine to alanine substitution at position 152 in the preprotein showed a marked increase in bioluminescence activity when compared to media containing the original secreted Renilla luciferase. This increase in light emission was due in part to enhanced stability of the mutant enzyme. This new enzyme represents a significant improvement in the sensitivity of the secreted Renilla luciferase assay for monitoring gene expression.     

The human fibroblast and human immune interferon genes and their expression in homologous and heterologous cells

The genetic information coding for human fibroblast interferon (IFN-beta) has been cloned both as a DNA copy (cDNA) and as a genomic clone. Human IFN-beta is made as a precursor and consists of a signal sequence 21 amino acid residues long followed by the mature protein 166 amino acids long. A single site for glycosylation is present. The human IFN-beta gene does not contain introns. Transfection of monkey cells with a chimeric SV40 derivative containing the human IFN-beta cDNA clone under control of the late SV40 promoter leads to secretion of high levels of IFN-beta. When a genomic clone is used in the same vector, IFN-beta synthesis can be further enhanced up to 30-fold by treatment with poly(rI) . poly(rC); this shows that a cis-active control element is present in the clone. An efficient expression system in Escherichia coli was worked out based on a plasmid containing the promoter PL of bacteriophage lambda, which is regulated by a temperature-sensitive repressor. This promoter is followed by a segment derived from bacteriophage MS2 that contains the ribosome-binding site of the replicase gene. The latter, however, is replaced by the human IFN-beta gene. Upon induction, high levels (about 5 x 10(9) IU 1(-1)) of IFN-beta are synthesized by the bacteria; this corresponds to about 2% of the total bacterial protein. The human immune (type II) interferon (IFN-gamma) gene has similarly been cloned. Partly purified mRNA derived from human spleen cells that had been induced with staphylococcal enterotoxin A was used as starting material. A full-length cDNA clone was sequenced. The total cDNA sequence is about 1150 nucleotides long; it contains a single open reading frame coding for 166 amino acids, the first 20 of which constitute the transmembrane signal. There are two sites for glycosylation. The amino acid sequence is quite different from that of IFN-alpha or IFN-beta, although a few similarities can be noted. The untranslated 3'-terminal region is about 550 nucleotides long. The IFN-gamma gene was expressed in monkey cells, again by using the SV40-derived vector, and the secreted product was characterized as true human IFN-gamma. A genomic clone in the form of a bacteriophage lambda derivative was also obtained. The IFN-gamma gene extends over at least 5 kilobases and contains at least two introns.     

Expression and production of recombinant human interleukin-2 in potato plants

Interleukin-2 is a pharmacologically important cytokine secreted by T lymphocytes. Recombinant interleukin-2 has been produced and found to be useful for many medical applications. Mass production of recombinant interleukin-2 will be prerequisite for a wider application of this molecule. In this study we investigated the possibility of using potato tubers for the production of recombinant human interleukin-2 in large quantity. A binary vector carrying the human interleukin-2 gene under a potato tuber-specific promoter (patatin promoter) was constructed. Several potato transformants expressing the human interleukin-2 gene were generated by Agrobacterium-mediated transformation. Expression of the human interleukin-2 gene was confirmed by Northern blotting and the protein level was determined by Western blot analyses. A bioassay revealed that human interleukin-2 expressed in the potato tuber supported proliferation of interleukin-2-dependent cells, CTLL-2. We found that the recombinant protein in the 2-week-old microtuber has the highest activity (115 units per gram of microtuber) and estimated that an average yield for a potato (average 200 g per potato) was 23,000 units of rhIL-2 activity. The results suggest that the potato tuber is an excellent system for the mass production of biologically active human interleukin-2.     

Isolation of an IgH gene circular DNA clone from human bone marrow.

Circular DNA was obtained from human bone marrow. Then a phage library was prepared and screened by use of two probes of the IgH gene; 5'-DHQ52, containing the 5' flanking region of DHQ52, and JH4.3, containing the sequence from JH3 to the 3' flanking region of JH6. One clone, HBMC-1, that was DHQ52+JH4.3- was obtained. HBMC-1 had the germline IgH region upstream of JH1 and the 3' flanking region of DXP1. A recombination signal sequence flanking the 5' side of the JH1 segment was attached to the recombination signal sequence flanking the 3' side of DXP1 forming a head-to-head structure of two 7mers with 10 nucleotides in-between. HBMC-1 is thus considered to be a circular DNA deleted as a consequence of DXP1-JH1 joining of the IgH gene.     

Expression of a cDNA encoding human 5-lipoxygenase under control of the STA1 promoter in Saccharomyces cerevisiae

A yeast-expression vector utilizing the STA1 promoter was constructed, and shown to be useful for the expression of a heterologous gene. A cDNA, encoding human 5-lipoxygenase (5LO), was inserted into the vector and expressed in Saccharomyces cerevisiae. The enzyme (yh5LO) produced in the transformant was purified to homogeneity from the cellular soluble fraction. The purified enzyme showed both 5LO and leukotriene A4 synthase activities, which were stimulated by Ca2+ and ATP. The N-terminal end of yh5LO contained five extra amino acids not present in 5LO purified from human leukocytes. A human 5LO-secretion vector containing the STA1 signal sequence was also constructed. When this hybrid gene was expressed in S. cerevisiae, its product was glycosylated and accumulated in the fractions related to the secretory pathway.     

Interleukin-2 promoter activation in T-cells expressing activated Ha-ras.

Antigen triggering of the T-cell receptor results in an accumulation of activated GTP-bound p21ras protein. To assess the role of ras protein in T-cell activation we have cotransfected the murine thymoma line EL4 with a construct capable of expressing a constitutively active, oncogenic form of Ha-ras and a reporter construct containing the human interleukin-2 promoter fused upstream of the bacterial gene for chloramphenicol acetyltransferase. We show that the ras oncoprotein contributes to interleukin-2 promoter activation. Its pattern of synergism with a calcium ionophore or the lymphokine interleukin-1 indicates that it replaces a signal mediated by protein kinase C. Interleukin-2 promoter activity in the presence of ras oncoprotein was inhibited by H7, a potent inhibitor of protein kinase C, but not by HA1004, an inhibitor of cyclic nucleotide-dependent kinase, suggesting that protein kinase C mediates the ras effect. In addition, we show that in these cells, expression of activated ras results in activation of a synthetic promoter containing several copies of an NF kappa B binding site.     

Cloning and analysis of the pseudogene for human epinephrine synthesizing enzyme, phenylethanolamine N-methyltransferase (PNMT)

1. This gene completely lacks the intervening sequences. 2. This gene is truncated at the 5' end peptide encoding region by 433 base pairs (bp). 3. The 502 bp of this gene containing poly(A) signal are completely identical to the 3' half of mRNA encoding region of functional gene. 4. This gene has a poly(A) tail and is flanked by direct repeat of 6 bp. 5. Here we report for the first time the complete sequence of a human pseudogene for phenylethanolamine N-methyltransferase and this is the first report of cloning of pseudogene for catecholamine biosynthetic enzymes.