A series of genes was generated from three interchangeable cassettes, each coding for a specific set of amino acids. The genes were inserted into two different fusion expression vectors and two direct expression vectors. The expression studies demonstrated that proteolytic stability of the proteins is affected by the N-terminal region of the protein.

Synthetic genes coding for artificial proteins with predefined and nutritionally valuable amino acid compositions have been constructed and cloned in bacterial plasmid vector pKK233-2. The genes were constructed from three easily interchangeable 'cassettes' encoding either essential, non-essential or branched-chain amino acid residues. A potential hairpin loop structure in the mRNA around the region of the ribosome binding site was probably the reason for blockage of translation from this vector. Two selected genes, AHB (containing one copy of each cassette) and A6 (consisting of six copies concatemerized A cassette) were cloned into pUR300, a beta-Gal fusion vector and expressed as fusion proteins beta-Gal-AHB and beta-Gal-A6.

The Mtv-8 associated provirus, GR40, is not expressed in vivo. However, upon transfection into rat XC cells, the two MMTV specific mRNAs of 35S and 24S are transcribed. Further, the level of these transcripts is augmented when the transfected cells are grown in the presence of dexamethasone (Ponta et al., 1983). No virus can be detected in the medium of the transfected cells. Intracellular protein analysis of these transfected cells shows that although apparently authentic gag proteins are synthesized, a novel protein of 68 kDa is the only env related protein detectable. The sequence of the env gene of GR40 was determined and the predicted amino acid sequence of the env protein obtained. A premature termination codon is present 68 amino acids before the COOH-terminus of the known MMTV env precursor Pr73env. This would result in an env protein of about 68 kDa, in agreement with the size of the env protein found in GR40 transfected cells. The lack of the carboxy terminus may be responsible for the non-processing of the aberrant env precursor protein, p68.

The proviral DNA of mouse mammary tumor virus (MMTV) contains a regulatory region closely associated with its promoter, which subjects transcription to the control of glucocorticoid hormones. Delimitation analysis of a chimeric MMTV long terminal repeat-thymidine kinase gene (LTR-tk) has shown that the hormonal regulation sequence is confined to 202 nucleotides preceding the LTR-specific RNA initiation site. A second RNA initiation site (tk-specific mRNA) placed close to the regulatory MMTV sequence by in vitro recombination is also subjected to hormonal stimulation in transfected cells. A series of plasmids with deletions around the LTR cap site progressing from 3' to 5' was made and functionally tested. In vitro deletion of MMTV LTR sequences comprising the RNA initiation sequence and the "TATA" box do not effect hormonal regulation at the tk-specific mRNA start site. Nucleotides up to position -59 from the LTR initiation site could be deleted without influence on the glucocorticoid regulation, whereas deletions to position -65 abolished the hormonal effect on the tk gene transcription. A short MMTV LTR segment containing nucleotides -236 to -52 from the LTR initiation site was recombined with the tk gene or the alpha-globin gene. This fragment confers hormonal inducibility onto the heterologous genes over distances of 0.4 or 1.1 kilobases. The hormonal response region functions when it is placed either 5' or 3' of the regulated gene in both of the possible orientations and is reminiscent of an enhancer sequence.

Glucocorticoids modulate various cellular functions such as proliferation, energy metabolism and thethesis of protein. In the present study, the response of collagen genes to dexamethasone in different stages of chick embryo development was studied in tendon and heart using Northern blot analysis and specific cDNA probes. The changes in collagen gene expression were compared to alterations in two reference mRNAs: actin and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The levels of specific mRNAs measured per ribosomal RNA in tendon and heart varied markedly during normal development. In tendon the relative levels of α1(I), α2(I) and α1(III) collagen mRNAs were highest between days 14–16 when also the synthesis of matrix proteins is most active. In heart the levels of these mRNAs peaked at day 12. In addition, qualitative differences were observed in the expression of actin genes between tendon and heart. Dexamethasonein high dose decreased collagen mRNA levels in tendons, while in heart a stimulatory effect was noted. Dexamethasone also decreased GAPDH mRNA levels in tendons. The alterations in gene expression after dexamethasone treatment in tendon and heart did not correlate with the level of specific glucocorticoid receptors, which varied markedly during the development of chick embryos. The cDNA for pro α1(I) collagen hybridized to two transcripts corresponding to 6.2 and 5.1 kb in tendon and heart. During normal development of chick embryos the ratio of 6.2/5.1 kb mRNAs decrease markedly in heart, but no such change was observed in tendons. Dexamethasone, however, decreased the ratio of 6.2/5.1 kb transcript in tendons. There was a significant correlation between the ratio 6.2/5.1 kb transcripts and total α1(I) mRNA both in tendon and heart, suggesting that the 6.2 kb transcript may be associated with the rate of synthesis of type I collagen.

In vitro manipulation of proviral DNA of mouse mammary tumour virus (MMTV) was used to construct mutants with defined deletions at the 5' end of the proviral gene. In the mutants 516, 1400 and 2000 nucleotides were removed from the 5' end. The deleted proviral DNA was tested for transcription and glucorticoid hormone regulation of viral RNA expression upon cotransfection into rat XC tk- cells with a thymidine kinase gene. Intact proviral DNA contained in the plasmid vector pBR322 and the deletion mutant pGR16 delta 516, missing 516 nucleotides of the 5' long terminal repeat (LTR) sequence, were transcribed in a hormone responsive fashion and produced RNA species of 35S and 24S. Deletion of the entire LTR sequence abolished MMTV transcription and the hormonal effect.

After transfection of mouse mammary tumor virus (MMTV) proviral DNA into cultured cells, the DNA is transcribed in a glucocorticoid-sensitive fashion. The large terminal repeat (LTR) region of MMTV is 1,328 nucleotides long and contains the regulatory information necessary for the hormonal response. We have constructed a MMTV LTR-thymidine kinase (tk) chimeric gene and have tested the biological activity of molecules containing various deletions in the LTR after transformation of LTK- APRT- mouse cells. In the TK+ transformants, both a LTR- tk chimeric RNA and an authentic tk RNA are correctly initiated and transcribed. The synthesis of the chimeric RNA as well as that of the tk RNA is hormonally regulated. A plasmid containing 202 nucleotides of LTR DNA 5' to the RNA initiation site is fully sensitive to glucocorticoids; 50 nucleotides still cause a residual inducibility.

The integrated DNA copies (proviruses) of RNA tumour virus genomes are flanked by long terminal repeat (LTR) sequences, which are generated by the duplication of segments present at both ends of the viral RNA1. LTRs vary in length from 330 (ref. 2) to 1,328 (this paper) base pairs (bp). The primary viral transcript starts in the left LTR (L-LTR) and terminates in the right LTR (R-LTR)3. We have now sequenced both LTRs from an inherited (endogenous) mouse mammary tumour virus (MMTV) of the GR strain. This provirus, which is one of five present in the haploid genome of GR mice4, does not produce virus5 and is not identical with the exogenous MMTV of GR mice6. The provirus (GR40) could nevertheless be transcribed and its transcription hormonally regulated after cloning and transfection into mouse L cells7. The LTRs are identical and exhibit the following features: a terminal inverted 6-bp repeat, a Goldberg–Hogness (TATAAA) sequence, sequences which have been implicated in polyadenylation and the termination of transcription, and an open reading frame. The open reading frame of the R-LTR starts at the env/LTR junction and encodes a 36,700-molecular weight (Mr) protein. The open reading frame of the L-LTR extends into the adjacent mouse sequence. A host sequence of 6 bp at the site of integration into the mouse chromosome is directly repeated.

A chimeric long terminal repeat-thymidine kinase (LTR-tk) gene has been used to define the sequence requirements for glucocorticoid induction of gene expression. The original LTR-tk gene contains an entire mouse mammary tumor virus (MMTV) LTR preceding the tk gene. This gene can be expressed in a hormone-responsive fashion upon transfection into L tk--cells to produce a chimeric LTR-tk mRNA. Stepwise deletion of nucleotide sequences 5' of the viral RNA initiation site revealed that 202 nucleotides upstream of the viral cap site are sufficient for the hormonal regulation. Deletion of 5' sequences up to 59 nucleotides upstream of the viral cap site abolished RNA initiation in the LTR and hormonal induction.

A recombinant lambda phage containing mouse mammary tumor virus (MMTV) proviral DNA was isolated from a gene library constructed from GR mouse liver DNA. Restriction enzyme analyses reveal that the cloned molecule contains a copy of one of the GR endogenous MMTV proviruses flanked on both sides by 2--3 kb of mouse genomic DNA. In this report we have examined the expression of the cloned MMTV provirus after cotransfection with the herpes thymidine kinase (TK; ATP:thymidine 5'-phosphotransferase,, EC 2.7.1.21) gene and integration into mouse LTK- cells. Nine individual TK+ transformants were selected, and all were found to contain MMTV-transfected DNA. One of the TK+ transformants was chosen for further study. Total poly(A)-containing RNA was isolated from the cells, and liquid hybridization analyses with MMTV cDNA showed that it contained 0.02% MMTV-specific RNA. The sizes of the MMTV-specific species were determined and found to correspond to the 35S and 24S mRNAs synthesized in MMTV-infected cells. Glucocorticoid hormones have been shown to increase the concentration of MMTV RNA in virus-infected cultured cells. Therefore, we tested the effect of dexamethasone on the concentration of MMTV-specific RNA in cells transfected with the MMTV proviral DNA. The amount of MMTV-specific poly(A)-containing RNA found in the cells grown in the presence of hormone was 0.17%. Therefore, dexamethasone causes an 8-fold increase in the amount of MMTV-specific RNA in mouse cells containing several copies of a cloned and transfected MMTV proviral gene.

The endogenous proviral copies of mouse mammary tumor virus (MMTV) were selected from a gene library of GR mouse DNA. We obtained five different lambda. MMTV recombinant clones. Four of them correspond to the 3' Eco RI fragments of the endogenous proviruses an one comprises an intact MMTV provirus with 2 to 3 kb of flanking mouse genomic DNA. Heteroduplex formation followed by S1 digestion under stringent conditions shows that there is nucleotide sequence heterology among the cloned endogenous proviral copies. Only one endogenous proviral copy, associated with the mtv-2 locus, was found to be totally homologous to the exogenous proviral DNA.