Elizabeth Lacy

The isolation of structural genes from libraries of eucaryotic DNA

We present a procedure for eucaryotic structural gene isolation which involves the construction and screening of cloned libraries of genomic DNA. Large random DNA fragments are joined to phage lambda vectors by using synthetic DNA linkers. The recombinant molecules are packaged into viable phage particles in vitro and amplified to establish a permanent library. We isolated structural genes together with their associated sequences from three libraries constructed from Drosophila, silkmoth and rabbit genomic DNA. In particular, we obtained a large number of phage recombinants bearing the chorion gene sequence from the silkmoth library and several independent clones of beta-globin genes from the rabbit library. Restriction mapping and hybridization studies reveal the presence of closely linked beta-globin genes.

Transformation of mammalian cells with genes from procaryotes and eucaryotes

Abstract We have stably transformed mammalian cells with precisely defined procaryotic and eucaryotic genes for which no selective criteria exist. The addition of a purified viral thymidine kinase (tk) gene to mouse cells lacking this enzyme results in the appearance of stable transformants which can be selected by their ability to grow in HAT. These biochemical transformants may represent a subpopulation of competent cells which are likely to integrate other unlinked genes at frequencies higher than the general population. Co-transformation experiments were therefore performed with the viral tk gene and bacteriophage ΦX174, plasmid pBR322 or the cloned chromosomal rabbit β-globin gene sequences. Tk + transformants were cloned and analyzed for co-transfer of additional DNA sequences by blot hybridization. In this manner, we have identified mouse cell lines which contain multiple copies of 4)X, pBR322 and the rabbit β-globin gene sequences. The ΦX co-transformants were studied in greatest detail. The frequency of co-transformation is high: 15 of 16 tk + transformants contain the ΦX sequences. Selective pressure was required to identify co-transformants. From one to more than fifty ΦX sequences are integrated into high molecular weight nuclear DNA isolated from independent clones. Analysis of subclones demonstrates that the ΦX genotype is stable through many generations in culture. This co-transformation system should allow the introduction and stable integration of virtually any defined gene into cultured cells. Ligation to either viral vectors or selectable biochemical markers is not required.

The structure and transcription of four linked rabbit β-like globin genes

Abstract Rabbit chromosomal DNA contains a cluster of four linked β-like globin genes arranged in the orientation 5′-β4-(8 kb)-β3-(5 kb)-β2-(7 kb)-β1–3′. Determination of the nucleotide sequence of gene β1 confirms that this gene corresponds to the second type of two common co-dominant alleles encoding the adult β-globin chain. With the exception of two nucleotide substitutions in the large intervening sequence (intron), the intron and flanking sequences are identical with the nucleotide sequence of the first type determined by Weissmann et al. (1979). A 14S polyadenylated transcript containing large intron sequences (possibly a mRNA precursor) is detected in the bone marrow cells of anemic rabbits. Gene β2 has limited sequence homology to adult and embryonic β-globin probes and lacks a detectable mRNA transcript in the erythropoietic tissues examined. It contains at least one intervening sequence analogous to the large intron in gene β1. Genes β3 and β4 both contain an intron of 0.8 kb. Partial DNA sequence analysis indicates that the large intron in β4 is located between codons for amino acids lysine and leucine in an analogous position to that of the large intron in β1. In addition, a second smaller intron interrupts the 5′ coding sequences of gene β4. Both genes β3 and β4 are transcribed in embryonic globin-producing cells. Their DNA sequence homology is limited, however, to a segment of approximately 0.2 kb located on the 5′ side of the large intron.

Analysis of DNA of isolated chromatin subunits

Partial digestion of rat liver nuclei with staphylococcal nuclease results in the liberation of nucleo-protein complexes consisting of one or more upsilon bodies. By velocity centrifugation we have isolated the monomeric subunit in relatively pure form. We find that this subunit contains 185 base pairs of DNA and 240,000 daltons of protein, resulting in a protein to DNA ratio identical to that of unperturbed chromatin. The isolated monomeric particle is further susceptible to internal nuclease attack resulting in the solubilization of 46% of the monomeric DNA. Analysis of the resistant DNA reveals a complex but highly reproducible pattern of DNA fragments ranging from 160 to 60 base pairs in length. Analysis of the reassociation kinetics of the isolated subunit DNA reveals that most, if not all genomic sequences, are involved in this basic subunit structure. No special frequency class of DNA is absent from upsilon bodies. Furthermore, virtually all liver sequences transcribed into mRNA are present in upsilon body DNA. These results indicate that upsilon body formation may be random with respect to DNA sequence and suggest that the mere presence of upsilon bodies over a specific region of DNA is not sufficient to restrict its transcription.

The linkage arrangement of four rabbit β-like globin genes

Abstract Four different regions of rabbit β-like globin gene sequences designated β1, β2, β3 and β4 were identified in a set of clones isolated from a bacteriophage λ library of chromosomal DNA fragments (Maniatis et al., 1978). Restriction mapping and blot hybridization (Southern, 1975) studies indicate that a subset of these clones containing β1 and β2 hybridizes to an adult β-globin cDNA clone (Maniatis et al., 1976) more efficiently than to a human γ-globin cDNA clone (Wilson et al., 1978), while another subset containing β3 and β4 displays the converse hybridization specificity. β1 was identified as the adult β-globin gene, while β2, β3 and β4 have not been identified with any known rabbit globin polypeptides. Cross-hybridization and transcriptional orientation experiments indicate that the set of β-like gene clones contains overlapping restriction fragments encompassing 44 kb of rabbit chromosomal DNA. In addition, all four genes have the same transcriptional orientation and are arranged in the order 5′-β4-β3-β2-β1–3′.

The nucleotide sequence of a rabbit β-globin pseudogene

Abstract We report the nucleotide sequence of a rabbit β-globin pseudogene, ψβ2. A comparison of the ψβ2 sequence with that of the rabbit adult β-globin gene, β1, reveals the presence of frameshift mutations and premature termination codons in the protein coding sequence which render ψβ2 unable to encode a functional β-globin polypeptide. ψβ2 contains two intervening sequences at the same locations in the globin protein coding sequence as β1 and all other sequenced β-globin genes. An examination of the DNA sequences at the intron/exon junctions suggests that a putative ψβ2 precursor mRNA could not be spliced normally. We compare the flanking and noncoding sequences of ψβ2 and β1 and discuss the evolutionary relationship between these two genes.