Globin evolution in the genus Xenopus: comparative analysis of cDNAs coding for adult globin polypeptides of Xenopus borealis and Xenopus tropicalis.

Globin mRNAs of Xenopus borealis and Xenopus tropicalis have been cloned and sequenced. The nucleotide and derived amino acid sequences were compared with each other and with already available data from Xenopus laevis. This analysis rendered clear evidence that the common ancestor of X. laevis and X. borealis, but not of X. tropicalis, had lost one amino acid of the beta-globins prior to a genome duplication event that preceded the segregation of the former two species. Replacement-site substitutions were used to calculate a rough time scale of genome duplication and species segregation. The results suggest an ancient separation between the X. laevis and the X. tropicalis groups occurring approximately 110-120 million years ago. Analysis of the amino acid chains demonstrated various alterations. However, some functional domains, like heme-binding sites and alpha 1 beta 2 contact sites, were subject to a high degree of conservation, indicating the existence of functional constraints on them also in the genus Xenopus.

Characterization of highly and moderately repetitive 500 bp Eco RI fragments from Xenopus laevis DNA.

Three different types of repetitive Eco RI fragments, which comigrate within a visible band of approximately 500 bp at gel electrophoresis of Xenopus laevis DNA Eco RI digests have been cloned and sequenced. These sequences are designated as Repetitive Eco RI Monomers: REM 1, REM 2 and REM 3. The sequences contain direct repeats, inverted repeats and palindromic elements. Genomic organization of the most abundant sequence (REM 1; 0.4% of total DNA) is that of an interspersed sequence. REM 2 (0.08%) is partly organized as an interspersed element and partly found in tandem arrangement, whereas REM 3 (0.02%) represents the tandemly repeated monomeric unit of a satellite DNA. In situ hybridization has shown that REM 1 and REM 2 sequences are found on most chromosomes, REM 1 being preferentially located on specific chromosomal loci. REM 3 is located near the centromere region of only one chromosome pair (presumably number 1). Hybridization of Northern blots from RNAs of different developmental stages revealed that REM 1, REM 2 and REM 3 sequences are transcribed and that transcription is under developmental control.

Satellite DNA from Xenopus laevis: comparative analysis of 745 and 1037 base pair Hind III tandem repeats.

Highly repetitive Hind III restriction fragments of 0.72-0.76 KBP from total Xenopus laevis genomic DNA are organized in a tandem like arrangement. Cloning of these fragments in pBR 322 with subsequent restriction site mapping and nucleotide sequence analysis of some selected clones showed two different types of sequences. 25-30% of material represent the oocyte specific 5 S DNA repeat units, 70-75% are similar to the recently described repeat elements of satellite 1 DNA. Hybridization of a genomic DNA library to such a 745 BP monomeric repeat unit and investigation of some clones with positive autoradiographic signals revealed structural heterogeneities of repeat elements, in that the 745 BP sequence cross-hybridized with 1037 BP Hind III repeat units. Nucleotide sequence analysis demonstrated that the two types of sequences show a homology of 84.3% and that the 1037 BP sequence additionally contains duplicated elements of the 745 BP sequence as well as apparently unrelated DNA sequences.

Quantitation of globin mRNA in individual human erythroblasts by in situ hybridization.

In situ hybridization of globin mRNA was performed in four human bone marrows using a 3H-cDNA derived from rat globin messenger. Erythroblasts were classified according to nuclear area as well as to morphological appearance after Giemsa staining. Grains over individual erythroblasts and reticulocytes were counted by incident light microphotometry. Reticulocytes from phenyl hydrazine-treated rats served as internal standards in order to allow a comparison of different experiments. The preliminary results from one normal bone marrow, one of stress erythropoiesis and two of aplastic anemia revealed the major content of globin mRNA in the states of basophilic erythroblasts or proerythroblasts. In all cases there was a tendency of decreasing globin mRNA content towards orthochromatic erythroblasts, and even more so, towards reticulocytes. The results show some differences between normal and diseased erythropoiesis which are under study on a larger scale now in order to ascertain their significance.