The complete cDNA sequence of mouse T200

P element transposition in Drosophila melanogaster is limited to the germ line because the third intron (the ORF2-ORF3 intron) of the P element transcript is spliced only in germ line cells. We describe a systematic search for P element sequences that are required to regulate the splicing of the ORF2-ORF3 intron. We have identified three adjacent mutations that abolish the germ line specificity and allow splicing of this intron in all tissues. These mutations define a 20-base regulatory region located in the exon, 12 to 31 bases from the 5' splice site. Our data show that this cis-acting regulatory sequence is required to inhibit the splicing of the ORF2-ORF3 intron in somatic cells.The splicing of pre-mRNA is one of the key regulatory events of gene expression in eukaryotic cells. Although much progress has been made toward understanding the biochemistry of splicing, little is known about its regulation. To further our understanding of this process, we are studying the germ line-specific splicing of the P element.P elements are transposable elements found in Drosophila melanogaster. They are the causal agents of P-M hybrid dysgenesis, a syndrome whose traits include high rates of sterility, mutation, and chromosomal rearrangement (12; for reviews see references 1, 5, and 7). P element transposition is tissue specific; transposition occurs at high rates in the germ line but is not detected in somatic tissue. Previously we have shown that the germ line specificity of P element transposition is due to germ line-specific splicing that joins open reading frame 2 (ORF2) to ORF3 (14). The first two introns of the P element are spliced in all tissues, but the ORF2-ORF3 intron is spliced only in the germ line, not in the soma (Fig. 1). cis-acting sequences must be present in the pre-mRNA which instruct the splicing machinery to splice the ORF2-ORF3 intron only in the germ line. The identification of these regulatory sequences may suggest a mechanism by which the germ line-specific splicing is regulated.Previously we have shown that a 240-base fragment that includes the third intron of the P element contains sufficient information to confer germ line specificity on the splice (15). For those experiments the ORF2-ORF3 intron of the P element was positioned between the Drosophila hsp70 (heat shock) promoter (20), plus about 1 kb of hsp70 proteincoding sequence, and the Escherichia coli lacZ gene, which encodes P-galactosidase (P-gal) (Fig. 2A). The hsp70 coding sequence was joined in frame to ORF2, and the lacZ gene was joined in frame following ORF3. For p-gal to be expressed from this construct, the hsp70 promoter must be activated and the ORF2-ORF3 intron must be spliced. The presence of P-gal activity in a heat-shocked fly containing this construct identifies tissues in which the ORF2-ORF3 intron is spliced. Because Transformants were also generated with the control construct hsp70-P(1911-[A2-3]-2183)1acZ, which differs from hsp70-P(1911-2183)-lacZ in that it contains the A2-3 deletion (a precise deletion of the ORF2...

Six a2-macroglobulin (a2M) cDNA clones were isolated from a human liver cDNA library by using synthetic oligonucleotides as hybridization probes. One of these, pa2M1, carries a 4.6-kilobase-pair insert, which was sequenced. The insert contains the coding sequences for the mature a2M polypeptide (1451 amino acids) and for a 23-amino acid signal peptide at the NH2 terminus of the precursor proa2M. At the 3' end of the insert a poly(A) addition signal A-A-T-A-A-A and part of the poly(A) tail of the messenger RNA were found. The protein sequence deduced from the nucleotide sequence agrees with the published a2M amino acid sequence for all except three residues. The a2M locus was assigned to human chromosome 12 by Southern blot analysis with DNA from a panel of mouse/human somatic cell hybrids, using a2M cDNA as a hybridization probe.a2-Macroglobulin (a2M) is a serum glycoprotein and a major plasma proteinase inhibitor with a wide specificity. a2M-related proteins are present in all vertebrate species (1-4). Human a2M is a tetramer of four identical 185-kDa subunits, arranged as a pair of dimers each consisting of two disulfidelinked monomers (5, 6). The a2M polypeptide has a so-called bait region and an internal thiol ester bond, which account for its properties as a proteinase inhibitor. The bait region, composed of a series of target peptide bonds for plasma proteinases (7, 8) (13,14).This suggests that the conformational change, which accompanies the complex formation between a2M and proteinases and the hydrolysis of the thiol ester bond, exposes regions of the a2M molecule that are recognized by these receptors. Receptor-mediated endocytosis of proteinase-a2M complexes by macrophages and liver cells leads to clearance of the complexes from the circulation.Internal thiol ester bonds are also found in the complement proteins C3 and C4 (15), which are derived from precursor polypeptides of size similar to a2M (180-200 kDa). Their thiol ester sites are found in positions comparable to those in a2M, and the amino acid sequences of all three thiol ester sites are conserved. These observations led to the proposal that the C3, C4, and a2M genes are derived from a common ancestral gene (16). The sequences of murine and human C3 and human C4 and partial cDNA sequences of murine C4 have recently been determined (17-21). Comparison of human a2M with murine C3 revealed a 25% overall sequence homology (17,18,22

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