A clone which contains the complete chicken ovalbumin gene, including its leader coding sequences, has been isolated. From electron microscopic analysis of this DNA we conclude that the minimal size of the transcriptional unit for ovalbumin is 7.7 kilobases. The DNA sequence of the region surrounding the 5' end of the ovalbumin gene is presented. Comparison of this sequence with those of other eukaryotic genes reveals striking similarities, possibly related to a promoter region, approximately 30 base pairs upstream from the site coding for the 5' end of the mRNA.

Double-stranded cDNA was synthesized from a mouse liver mRNA fraction enriched for metallothionein mRNA activity, ligated to restriction site linkers, inserted into pBR322, and used to transform Escherichia coli 1776. The sequence of the largest plasmid containing DNA that hybridized to metallothionein mRNA was determined and shown to contain a 38base-pair insert that includes the entire coding region and 3' untranslated region of metallothionein-I. The We became interested in studying MT gene regulation because the amount of translatable MT mRNA increases rapidly in response to heavy metals or glucocorticoids (3, 4). MT synthesis occurs predominantly in liver and kidney in vivo, but nearly all cell types are responsive in culture (unpublished observations; refs. 5 and 6). In addition, cell cultures can be made resistant to Cd, and these lines overproduce MT (7, 8). The molecular mechanisms by which heavy metals and steroids regulate MT mRNA production are unknown. Because an understanding of gene regulation depends upon specific probes for RNA and DNA sequences, we have initiated our investigation by isolating a MT-I cDNA clone and using it to select genomic clones containing the MT-I gene.MATERIALS AND METHODS Preparation of RNA. Swiss Webster mice (25 g) were injected subcutaneously with 1.5 ,umol of CdSO4 and 0.5 ,mol of ZnSO4. Six hours later, a 5% (wt/vol) liver homogenate was prepared in NaDodSO4 and proteinase K, and total nucleic acids were extracted. RNA was precipitated with 2 M LiCl; poly(A)-RNA was selected by oligo(dT)-cellulose chromatography and sedimented for 20 hr at 280,000 X g on 12-ml, 5-20% sucrose gradients in 10 mM Hepes (pH 7.5) (9). Fractions (0.5 ml) were collected and assayed directly by translation; those containing MT mRNA were pooled and sedimented on a second, identical gradient. Fractions were assayed for MT mRNA activity with a nuclease-treated rabbit reticulocyte lysate essentially as described (10) except that no exogenous amino acids other than [85S]cysteine (400 Ci/mmol; 1 Ci = 3.7 X 10"0 becquerels) were added and the reaction mixtures contained 5 mM dithiothreitol. After translation, samples (5 ,l) were incubated with 20,Ml of 100 mM iodoacetate in 0.5 M Tris-HCl (pH 8.8) for 1 hr at 37°C, electrophoresed on a NaDodSO4/polyacrylamide gel for 14 hr at 10 mA, and analyzed by fluorography. Purified mouse MT (11) was included as a standard.Preparation of Double-Stranded (ds) cDNA, Insertion into pBR322, and Transformation of X1776. Double-stranded cDNA was prepared (12) and ligated to a 1:1 mixture of HindIII and EcoRI linkers (Collaborative Research, Waltham, MA) prior to being inserted into EcoRI/HindIII-digested pBR322 as described (13). Transformation of E. coli X1776 was performed as described (14) with the following modifications: (i) the 100 mM CaCl2 buffer was replaced by 70 mM MnCl2/30 mM CaCl2/40 mM NaOAc, pH 5.6; (ii) the cells were transformed with plasmid at a concentration of 3 ,ug/ml; and (iii) the transformed cells were incubated at 37°C in L broth f...

The conalbumin gene has been cloned and shown to consist of at least 17 exons approximately 60-200 base pairs long. The DNA sequence upstream from the region coding for the 5' end of the mRNA shows similarities with sequences present in homologous positions in other genes. High and low frequency repetitive sequences are found both upstream from the conalbumin gene and within one intron.