As shown by Southern blot analysis, the metallothionein-1 (MT-1) genes in rats comprise a multigene family. We present the sequence of the MT-1 structural gene and compare its features with other metaliothionein genes. Three MT-1 pseudogenes which we sequenced apparently arose by reverse transcription of processed mRNA transcripts. Two of these, MT-1*a and MT-l*c, are retrogenes which derive from the MT-1 mRNA, having diverged from the MT-1 gene 6.9 and 2.6 million years ago, respectively. The third, MT-l*b, differs from the MT-1 cDNA by only three nucleotide alterations. Surprisingly, MT-l*b also preserves sequence homology for 142 base pairs 5' to the transcription initiation site of the parent gene; it contains a promoter sequence sufficient for specifying metal ion induction. We identified, by Si nuclease mapping, an RNA polymerase II initiation site 432 base pairs 5' of the MT-1 transcription initiation site of the MT-1 structural gene which could explain the formation of the mRNA precursor to this pseudogene. We were unable to detect MT-i*b transcripts, either in liver tissue or after transfection. We conclude that the absence of detectable transcripts from this pseudogene is due to either a reduced level of transcription or the formation of unstable transcripts as a consequence of the lack of a consensus sequence normally found 3' of transcription termination in the MT-1 structural gene.Metallothioneins are 6,000-dalton, cysteine-rich proteins that bind the metal ions zinc, copper, cadmium, and mercury (see 32 for review). Metallothioneins are usually isolated as two major isoforms, designated MT-1 and MT-2. The metallothionein (MT) genes are inducible by metal ions (4, 17), glucocorticoid hormones (19,20,36,37), stress (51), bacterial endotoxin (16), iodoacetate (18), and interferon (22). Human MT-1 and MT-2 genes show differential expression with metal ion and hormone inducers (57). The proposed functional roles of metallothioneins include: protection against heavy metal ion intoxication by cadmium or mercury (49); biological response to stress (51,64), and regulation of zinc and copper during development (3,5,52,59). The existence of numerous independent induction responses to different agents regulating MT gene expression suggests a complex role for the regulatory recognition sequences in these genes (16,22,30,43). The sequencing of members of a family of MT genes allowed us both to explore in detail the evolutionary relationship among these genes and to compare the regions of sequence involved in regulating MT gene expression. MATERIALS AND METHODSScreening of Charon 4a library, DNA sequence analysis, and computer analysis of sequence data. The screening of a rat liver Charon 4a genomic library for MT-1 sequences and the restriction maps for the inserts in p34 (MT-1 structural gene), p21 (MT-ltja), p27 (MT-lhb), and p5 (MT-14c) have already been reported (2). The sequence analysis of these clones was done by a combination of base-specific chemical cleavage of 5' end-labeled restriction fragments from...