The Structure, Expression and Chromosomal Arrangement of Rabbit β Globin Genes

Citation

Lacy, Elizabeth Hardy (1981) The Structure, Expression and Chromosomal Arrangement of Rabbit β Globin Genes. Dissertation (Ph.D.), California Institute of Technology. doi:10.7907/z3xt-k222. https://resolver.caltech.edu/CaltechTHESIS:09212021-231109296

Abstract

Rabbits contain at least three β-like and two α-like globin polypeptides which are differentially synthesized during embryonic development. The structure, expression and chromosomal arrangement of the gene family encoding the β-like polypeptides is described.

A novel procedure was used to isolate the rabbit β globin gene family which does not require the partial purification of single copy genes. Large random rabbit genomic DNA fragments were joined to phage lambda vectors by using synthetic DNA linkers. The resulting recombinant lambda-rabbit DNA molecules were packaged in vitro into viable phage particles and amplified to produce a permanent library of rabbit genomic sequences. The library was screened using cloned globin cDNA probes and an in situ plaque hybridization procedure.

From a screen of the rabbit library, nine clones were isolated which contain four different β-like gene sequences (β1, β2, β3 and β4). Restriction enzyme mapping and blot-hybridization studies indicated that the nine clones contain overlapping restriction fragments, which together encompass 44 kilobase pairs (kb) of contiguous rabbit chromosomal DNA. Therefore, the four rabbit β-like globin genes are physically linked. In addition, all four genes are transcribed off the same strand of DNA in the orientation 5'-β4-β3-β2-β1-3'. A combination of blot-hybridization, R-looping, and DNA sequencing experiments demonstrated the presence of a large intervening sequence in all four genes and a second, smaller intervening sequence in β1, β2 and β4.

Determination of the nucleotide sequence of β1 showed that this gene codes for the second type of two common codominant alleles encoding the rabbit adult β globin chain. A presumptive 1450 nt polyadenylated precursor to β1 mRNA was detected in adult bone marrow. Mature mRNA transcripts from genes β3 and β4 were found in rabbit embryonic erythroid cells, thereby identifying β3 and β4 as embryonic and/or fetal β-like glob in genes. RNA blotting experiments confirmed that genes β1, β3 and β4 are differentially expressed during development.

No β2 transcripts were identified in anemic adult bone marrow or reticulocytes or in 12-day rabbit embryos. A DNA sequence analysis demonstrated that β2 cannot code for a functional β globin polypeptide and is therefore a globin pseudogene.

The mRNA expression of β1 was analyzed in a heterologous host cell using DNA-mediated transformations. Thymidine kinase (tk) minus mouse L cells were cotransformed with a lambda clone containing a chromosomal copy of the rabbit adult β globin gene, β1, and the herpes virus tk gene. The tk⁺ transformants containing copies of the rabbit gene were analyzed for rabbit β globin transcripts. One transformant was found to contain 5 copies per cell of a cytoplasmic 9S polyadenylated rabbit β globin transcript. Berk-Sharp S1 experiments demonstrated that both intervening sequences are spliced out precisely from the rabbit 9S β globin transcripts in the transformed mouse L cells. This result indicates that RNA splicing mechanisms are not species-specific. However, the 5' termini do not contain 48 ± 5 nucleotides present in the β globin mRNA of rabbit reticulocytes. Therefore, the initiation and/or 5' processing of rabbit β globin nuclear RNA precursors are not identical in transformed mouse fibroblasts and rabbit reticulocytes.

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