Insulin receptor substrate-1 (IRS-1) is the major substrate of insulin receptor and IGF-1 receptor tyrosine kinases; it has an apparent relative molecular mass of 160-190,000 (M(r), 160-190K) on SDS polyacrylamide gel. Tyrosine-phosphorylated IRS-1 binds the 85K subunit of phosphatidylinositol 3-kinase which may be involved in the translocation of glucose transporters and the abundant src homology protein (ASH)/Grb2 which may be involved in activation of p21ras and MAP kinase cascade. IRS-1 also has binding sites for Syp and Nck and other src homology 2 (SH2) signalling molecules. To clarify the physiological roles of IRS-1 in vivo, we made mice with a targeted disruption of the IRS-1 gene locus. Mice homozygous for targeted disruption of the IRS-1 gene were born alive but were retarded in embryonal and postnatal growth. They also had resistance to the glucose-lowering effects of insulin, IGF-1 and IGF-2. These data suggest the existence of both IRS-1-dependent and IRS-1-independent pathways for signal transduction of insulin and IGFs.

The c-myb protooncogene encodes a sequence-specific DNA-binding protein (c-Myb) that induces transcriptional activation or repression. We have identified three functional domains of the mouse c-Myb protein that are responsible for DNA binding, transcriptional activation, and negative regulation, respectively. In addition to the DNAbinding domain, which is located near the N terminus, an adjacent region (the transcriptional activation domain) containing about 80 amino acids was found to be essential for transcriptional activation. Deletion of a region spanning about 175 amino acids of the C-proximal portion increased transcriptional activation markedly,-revealing that this domain normally represses activation. Differences between the transcriptional activation and repression functions of c-Myb and v-Myb are discussed in the light of these functional domains. Our results suggest that transcriptional activation may be involved in transformation by myb gene products. When the sequences of the MBS-I and MBS-II sites were compared, 11 of 19 base pairs (bp) were identical. MBS-I is a high-affinity site and was shown to be a c-Myb-dependent enhancer element. MBS-II is a low-affinity site, and tandem repeats of the sequence containing this site induce cMyb-dependent transcriptional repression (unpublished results). Here we report the identification of three functional domains of c-Myb: a DNA-binding domain, a transcriptional activation domain, and a negative regulatory domain. The functional differences between c-Myb and v-Myb are discussed in the light of the structures of these functional domains.MATERIALS AND METHODS Plasmid Construction. The effector plasmids pact-c-myb, in which the 5' regulatory region of the chicken cytoplasmic 13-actin gene is linked to the mouse c-myb gene, and pactl, which was constructed by deletion of the c-myb sequence from pact-c-myb, have been described (14). The reporter plasmids pMFcolCAT6MBS-I and pMFcolCAT6SV-II contain six tandem repeats of the MBS-I and the SV-II sequence, respectively, in the BamHI site of the plasmid pMFcolCAT, in which the bacterial chloramphenicol acetyltransferase (CAT) gene is linked to the mouse a2(I)-collagen promoter. The SV-II sequence contains the MBS-II site and corresponds to positions 184-218 in the SV40 genome. All plasmids designed to express mutant c-Myb proteins in cultured cells were generated from the plasmid pact-c-myb. To make the CT1, CT5, CT6, and CT7 mutants, termination codons were introduced at nucleotides 1537, 616, 463, and 307, respectively, by site-specific mutagenesis as described by Kunkel et al. (15). Nucleotide numbers are as in ref. 5. To obtain the NT1, NT3, and NT4 mutants, the sequence recognized by the restriction enzyme Nco I was introduced at nucleotides 147, 2%, and 455, respectively, by site-specific mutagenesis, and the regions between the introduced Nco I sites and the Nco I site at nucleotide 36 that overlaps the normal c-myb initiation codon were deleted. In-frame deletion mutants ADB, ANR, and ATA were construc...

Diabetes mellitus associated with the A-->G mutation at position 3243 of mitochondrial leucine transfer RNA represents a subtype of diabetes found in both patients with IDDM and patients with NIDDM in Japan.

By screening a lambda gt11 library with the multimerized sequence of the cAMP response element (CRE), we isolated human clones encoding the CRE binding protein, CRE‐BP1, from a human brain cDNA library. CRE‐BP1 expressed in Escherichia coli bound not only to the CRE element of the somatostatin and fibronectin genes, but also to the CRE element of the adenovirus E4 gene, suggesting that the protein was not distinguishable from the adenovirus transcription factor, ATF. The human CRE‐BP1 clone encoded a 54.5 kd protein similar at its carboxy terminus to the leucine zipper motifs found in other enhancer binding proteins such as C/EBP and c‐jun/AP‐1. CRE‐BP1 mRNA was expressed in all of the cells examined and was abundant in brain. The structure of CRE‐BP1 and its recognition elements suggest that cellular response to extracellular stimuli is controlled by a family of transcription factors that bind to related cis‐active elements and that contain several highly conserved domains.