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A promoter‐deletion derivative of the spinach trnM2 gene was used for the identification and characterization of the promoter regions for the spinach chloroplast RuBisCo large subunit (rbcL), ATPase beta‐subunit (atpB) and QB‐polypeptide (psbA) genes. The DNA sequences 5′ upstream from the transcriptional start sites of these genes share homology with the ctp1 and ctp2 arrangement found for the trnM2 transcription unit and the canonical Escherichia coli ‘‐10′ and ’‐35′ promoter regions. Synthetic DNA fragments of approximately 40‐bp regions, including the defined transcriptional start sites and proximal residues, from rbcL, atpB and psbA, were fused to the trnM2 deletion mutant 51. The promoter‐fusion constructs direct the correct transcription of tRNAMet2 in the chloroplast extract with distinct efficiencies. The ctp1‐ and ctp2‐like elements in the trnM2, rbcL and psbA promoter regions can be interchanged to yield functional chimeric promoters of varying strengths. As a result, ctp1 sequences from atpB and psbA, trnM2 and rbcL, respectively, can be ordered TTGACA greater than TTGCTT greater than TTGCGC with respect to their intrinsic strengths. Single base pair changes were introduced into the ctp2‐like element in the psbA promoter region. In analogy to similar base pair changes which lower promoter efficiency in E. coli, these mutations result in reduced transcription levels in the chloroplast extract. The data are consistent with a prokaryotic model for chloroplast promoter function.

The function of two alpha‐helical regions of mouse interleukin‐2 were analyzed by saturation substitution analysis. The functional parts of the first alpha‐helix (A) was defined as residues 31‐39 by the observation that proline substitutions within this region inactivate the protein. Four residues within alpha‐helix A, Leu31, Asp34, Leu35 and Leu38, were found to be crucial for biological activity. Structural modeling suggested that these four residues are clustered on one face of alpha‐helix A. Residues 31 and 35 had to remain hydrophobic for the molecule to be functional. At residue 38 there was a preference for hydrophobic side chain residues, while at residue 34 some small side chain residues as well as acidic or amide side chain residues were functionally acceptable. Inactivating changes at residue 34 had no effect upon the ability of the protein to interact with the p55 receptor. Disruption of the fifth alpha‐helix (E), which had little effect upon biological activity, resulted in an inability of the protein to interact with the p55 receptor. Mutagenesis of the alpha‐helix E region demonstrated that alpha‐helicity and the nature of the side chain residues in this region were unimportant for biological activity. The region immediately proximal to alpha‐helix E was important only for the single intramolecular disulfide linkage.

We have analyzed structure–function relationships of the protein hormone murine interleukin 2 by fine structural deletion mapping. A total of 130 deletion mutant proteins, together with some substitution and insertion mutant proteins, was expressed in Escherichia coli and analyzed for their ability to sustain the proliferation of a cloned murine T cell line. This analysis has permitted a functional map of the protein to be drawn and classifies five segments of the protein, which together contain 48% of the sequence, as unessential to the biological activity of the protein. A further 26% of the protein is classified as important, but not crucial, for the activity. Three regions, consisting of amino acids 32‐35, 66‐77 and 119‐141 contain the remaining 26% of the protein and are critical to the biological activity of the protein. The functional map is discussed in the context of the possible role of the identified critical regions in the structure of the hormone and its binding to the interleukin 2 receptor complex.

A transcription extract from purified spinach chloroplast was used to test chloroplast DNA sequences for their function as promoter elements. Chloroplast tRNA genes are correctly transcribed in the extract by a soluble RNA polymerase, and precursor molecules are processed into mature tRNAs. Transcription of the spinach chloroplast tRNA2Met gene (trnM2) in vitro requires 5′ upstream DNA sequences. Deletion of 5′ DNA sequences with exonuclease Bal31 was used to establish the 5′ boundary of the promoter region. This boundary is part of a DNA sequence with partial homology to the prokaryotic ‐35 region. Seventeen base pairs downstream from this sequence a DNA sequence occurs which is homologous to the prokaryotic ‐10 region. We used synthetic oligonucleotides fused to trnM2 5′ deletion mutants to create insertions, deletions and base substitutions in these regions. Internal deletion mutants demonstrated that the ‐10 promoter element is also required for transcription in vitro. The arrangement of DNA sequences recognised by the chloroplast RNA polymerase resembles the prokaryotic promoter organization.

We describe a new cystatin in both mice and humans, which we termed leukocystatin. This protein has all the features of a Class II secreted inhibitory cystatin but contains lysine residues in the normally hydrophobic binding regions. As determined by cDNA library Southern blots, this cystatin is expressed selectively in hematopoietic cells, although fine details of the distribution among these cell types differ between the human and mouse mRNAs. In addition, we have determined the genomic organization of mouse leukocystatin, and we found that in contrast to most cystatins, the leukocystatin gene contains three introns. The recombinant proteins corresponding to these cystatins were expressed in Escherichia coli as N-terminal glutathione S-transferase or FLAGTM fusions, and studies showed that they inhibited papain and cathepsin L but with affinities lower than other cystatins. The unique features of leukocystatin suggests that this cystatin plays a role in immune regulation through inhibition of a unique target in the hematopoietic system.

The evolution of the group II intron in the plastid gene encoding tRNA(Val)UAC (trnV) from seven plant taxa was studied by aligning secondary and other structural features. Levels of evolutionary divergence between six angiosperms and a liverwort, Marchantia polymorpha, were compared for the six domains commonly demonstrated for group II introns and were shown to be statistically heterogeneous. Evolutionary rates varied substantially among various domains and other features. Domain II showed the highest evolutionary rate, approaching the synonymous substitution rate reported for cpDNA-encoded genes, while domain VI and the helix and loop region bearing EBS1 evolved at rates similar to those for nonsynonymous substitutions of a number of cpDNA-encoded genes. The minimum free-energy structure of domain I varied among the seven taxa, suggesting that possible protein-RNA or tertiary interactions are important for intron processing.

A 2.1-kilobase Bgl II DNA fragment from Escherichia coli containing livK, the gene coding for the leucine-specific binding protein, has been cloned into the BamHI site of the plasmid vector pBR322. The DNA sequence of segments of the resulting plasmid, pOX7, established the location of the livK gene and the direction of its transcription. In vitro protein synthesis directed by pOX7DNA yielded the Mr 42,000 precursor of the leucine-specific binding protein and a small amount of the Mr 39,000 mature protein. Continued incubation of the in vitro reaction mixture after DNase and RNase treatment resulted in additional processing. The DNA sequence of the beginning of livK suggested that 23 additional amino acid residues are present as an extension of the NH2 terminus of the mature protein. Amino acid sequence analysis established that the precursor has the predicted 23-residue extension. Proteolytic digestion studies with the precursor and mature forms of the leucine-specific binding protein indicate that there are conformational differences between the two. This suggests a possible role for the signal sequence in determining the conformation of the binding protein precursor that is recognized by the membrane.

Isolation of pea lectin from immature seeds, together with in vivo pulse-chase labeling and in vitro translation of RNA from such seeds, all gave results which indicated strongly that pea lectin is initially synthesized as a large precursor (pre-pro-form) (Mr = 25,000) which is cleaved both co- and post-translationally to yield the alpha and beta subunits of mature lectin. Two overlapping cDNA plasmids complementary to lectin mRNA were sequenced. They coded for both beta and alpha subunits of lectin, in the orientation beta leads to alpha from the NH2 terminus, together with a hydrophobic NH2-terminal leader sequence and included a 3' untranslated region of 124 nucleotides. In the appropriate reading frame, no stop codon was found between the coding sequences for the beta and alpha subunits, confirming the synthesis of pea lectin via a pre-pro-form. The entire pea lectin sequence showed a high degree of homology with the published amino acid sequences of lectins from lentil, broad bean, and, to a lesser extent, with concanavalin A from jack bean. Sequence variation occurred mainly in those regions thought not to be involved in metal- and sugar-binding or in the formation of beta-pleated sheets.

Functional receptors for interleukin (IL)-4 and IL-13 on endothelial cells consist of the 130-kDa IL-4 receptor alpha-chain (IL-4Ralpha) and a 65-75-kDa IL-13 binding subunit that are expressed in a ratio of about 1:3, respectively. The restricted number of IL-4Ralpha limits subunit heterodimerization and in turn receptor-mediated signaling. We report here, the effects of tumor necrosis factor alpha (TNF-alpha) on the expression of the receptor subunits for IL-4 and IL-13. By flow cytofluorometry and receptor-binding analysis of iodinated IL-4 and IL-13, stimulation with TNF-alpha-induced a 2-3-fold increase of the IL-4Ralpha expression. The up-regulation was also confirmed at the transcriptional level by reverse transcription-polymerase chain reaction. Radioligand cross-linking experiments revealed no change in the subunit composition of the TNF-alpha-induced receptor complex. Nevertheless, TNF-alpha stimulation led to increased activation of the IL-4-specific signal transducers and activators of transcription protein (Stat6) by IL-4 and IL-13. Thus, TNF-alpha corrects the subunit imbalance of the endothelial IL-4.IL-13 receptor complex thereby increasing receptor heterodimerization and in turn the signaling capability by IL-4 and IL-13.

The four genes encoding the components of the high-affinity branched-chain amino acid transport systems in Escherichia coli (livH, livG, livJ, and livK) have been cloned into lambda phage and subsequently into the plasmid vector pACYC184. The presence of the four structural genes and their accompanying regulatory regions on the resultant plasmid, pOXI, was confirmed by genetic complementation and analysis and by transport studies carried out on the appropriate transformed mutant strains. When pOX1 DNA was used to direct an in vitro transcription/translation system, four major polypeptide products were produced. Immunoprecipitation with antibody directed against the LIV-binding protein identified the two leucine-binding proteins as products of in vitro synthesis. The binding proteins were produced in precursor forms and had molecular weights approximately 2500 higher than the processed, mature forms. A minicell-producing strain transformed with plasmid pOX1 produced the binding proteins in the processed form.

The DNA sequence of the 570 base pairs that precedes and includes the beginning of the ilvE gene shows no evidence of a leader-attenuator region. Instead, the sequence shows that the ilvE gene is preceded by another structural gene, presumably the normally cryptic ilvG gene. In vitro transcription of the two plasmids (pLC26-3 and pRL5) containing the regulatory region of the ilvEDA operon results in the formation of two "leader" RNAs. These results are consistent with the suggestion that the ilvEDA operon is regulated by an attenuator mechanism and that the attenuator region lies before the ilvG gene.

Interleukin (IL)-13 elicits a subset of the biological activities of the related IL-4. The basis of this functional similarity is that their specific cell-surface receptors (called IL-13R and IL-4R) are distinct, yet are complex and share a common subunit(s). The IL-4R primary binding subunit (called IL-4R alpha) does not by itself bind IL-13. We show that the ability of IL-13 to partially compete for IL-4 binding to some human cell types depended on co-expression of IL-4R and IL-13R. However, IL-13 binding was always associated with IL-4 binding. Hyper-expression of IL-4R alpha on cells expressing both IL-4R and IL-13R decreased their binding affinity for IL-4, abrogated the ability of IL-13 to compete for IL-4 binding, and yet had no effect on IL-13R properties. Anti-human IL-4R alpha monoclonal antibodies which blocked the biological function and binding of IL-4 also blocked the function and binding of IL-13. These data show that IL-4R alpha is a secondary component of IL-13R.