Potential immunocompetence of proteolytic fragments produced by proteasomes before evolution of the vertebrate immune system.

To generate peptides for presentation by major histocompatibility complex (MHC) class I molecules to T lymphocytes, the immune system of vertebrates has recruited the proteasomes, phylogenetically ancient multicatalytic high molecular weight endoproteases. We have previously shown that many of the proteolytic fragments generated by vertebrate proteasomes have structural features in common with peptides eluted from MHC class I molecules, suggesting that many MHC class I ligands are direct products of proteasomal proteolysis. Here, we report that the processing of polypeptides by proteasomes is conserved in evolution, not only among vertebrate species, but including invertebrate eukaryotes such as insects and yeast. Unexpectedly, we found that several high copy ligands of MHC class I molecules, in particular, self-ligands, are major products in digests of source polypeptides by invertebrate proteasomes. Moreover, many major dual cleavage peptides produced by invertebrate proteasomes have the length and the NH2 and COOH termini preferred by MHC class I. Thus, the ability of proteasomes to generate potentially immunocompetent peptides evolved well before the vertebrate immune system. We demonstrate with polypeptide substrates that interferon gamma induction in vivo or addition of recombinant proteasome activator 28alpha in vitro alters proteasomal proteolysis in such a way that the generation of peptides with the structural features of MHC class I ligands is optimized. However, these changes are quantitative and do not confer qualitatively novel characteristics to proteasomal proteolysis. The data suggest that proteasomes may have influenced the evolution of MHC class I molecules.

Deletion of the carboxyl-terminal transactivation domain of MGF-Stat5 results in sustained DNA binding and a dominant negative phenotype.

The Stat (signal transducer and activator of transcription) factors transmit cytokine, growth factor, and hormone responses. Seven members of the Stat gene family are known. MGF-Stat5a has been discovered as a mediator of the prolactin response in mammary epithelial cells. Two closely related variants of Stat5, Stat5a and Stat5b, are encoded by distinct genes. We examined the functional properties of the carboxyl termini of these molecules. Wild-type Stat5a (794 amino acids) and the carboxyl-terminal deletion mutant Stat5a delta 772 supported prolactin-induced transcription of a beta-casein promoter-reporter construct in COS7 cells; Stat5a delta 750 did not. Upon prolactin activation, tyrosine phosphorylation and the specificity of DNA binding were indistinguishable among the three Stat5a variants. Tyrosine dephosphorylation and the downregulation of the DNA-binding activity were delayed in the Stat5a delta 750 mutant. The carboxyl-terminal transactivation domain of Stat5a, amino acids 722 to 794, can be conferred to the DNA-binding domain of the yeast transcription factor GAL4. Coexpression of Stat5a or Stat5b and of the carboxyl-terminal deletion mutants resulted in the suppression of transcriptional induction in COS or Ba/F3 cells. We propose that Stat5a delta 750 and Stat5b delta 754 are lacking functional transactivation domains and exert their dominant negative effects by blocking the DNA-binding site in Stat5-responsive gene promoters.

Evolution of the chAB4 multisequence family in primates.

Approximately 50 members of the primate-specific multisequence family chAB4 are located as clusters at eight different chromosomal loci within the human genome. The whole cloned region of chAB4 represents a single-copy or low-copy sequence in all nonhuman primates tested, with the exception of the chimpanzee, for which we found chAB4 copy numbers similar to those in the human. An Alu element was inserted into chAB4 after the divergence of the Old World monkeys from the hominoids but before chAB4 was amplified. The first amplification step could be dated after the great apes and the human diverged from the Old World monkeys. We have evidence that neither the copy numbers nor the chromosomal locations remained stable after this initial step and that gross alterations in the relative copy numbers of individual family members occurred even after the divergence of the human and the chimpanzee. Taken together, our data suggest that chAB4, in an evolutionary sense, is an unusually unstable sequence family.

SQUIRREL: Sequence QUery, Information Retrieval and REporting Library. A program package for analyzing signals in nucleic acid sequences for the VAX.

A computer tool is described for comparison, analysis and search of genetic signals. The method is based on sequence consensus matrices. It assumes that a genetic signal (such as a promoter, enhancer or whatever) is composed of several signal blocks separated from each other by variable distances. A set of programs is presented to perform the analysis. The result of such an analysis is a description of the investigated signal including matrices for each signal block, distances between each block and distribution of the values. Programs are provided to search for a signal using results from previous analysis. The method is able to align large sets of sequences within a few minutes and to check the quality of the alignment. An analysis of E.coli promoters is provided as an example.

A menu-shell for the GCG programs.

We provide a menu-driven integration of the genetic programs of the Genetics Computer Group (GCG). This allows in-experienced users a very simple access to all GCG programs regardless of the system environment. No modifications to the GCG package are necessary.

COOL--a VAX program for finding COmmon OLigomers in nucleic acid sequences. Thyroid hormone receptor sequences used as an example.

COOL is a program designed to find COmmon OLigomers in a number of nucleic acid sequences. The results of COOL serve as starting points in sequence analysis investigations as well as suggestions for polymerase chain reaction probes. As an example we analyzed thyroid hormone receptor genes and found two oligomers which are characteristic of almost all those genes.