ABSTRACT
Nicotiana tabacum var. Carlson protoplast culture conditions were modified to contain a cell wall inhibitor, 2,6-dichlorobenzonitrile, in order to delay cell wall regeneration and to allow efficient nuclear and cytoplasmic microinjections. Under modified conditions, the protoplast preparations appeared healthier as compared to the control protoplasts and showed no resistance at all during microinjection. Furthermore, the duration of protoplast microinjection was extended for up to 3-4 days. In order to set up nuclear microinjections, the nuclei of these protoplasts were stained either before or after immobilization without any adverse effect on their mitotic activity. Successful cytoplasmic microinjections were demonstrated by injecting Alfalfa mosaic virus (AMV) RNA, which resulted in viral infection of 14% of the injected protoplasts.
ABSTRACT
As an initial step in the investigation of the structure, evolution and developmental regulation of the glycogen phosphorylase gene family, we have isolated partial cDNAs to rat, rabbit and human muscle phosphorylase mRNAs. Sequence comparisons of these cDNAs in regions that encode portions of the enzyme located near and encompassing the C terminus show that there is a high degree of interspecies conservation of structure in this region. Conservation of amino acid and nucleotide sequence is high, approximately 96% and 90% homology, respectively, among all three species. In addition, most of the amino acid changes that have occurred conserve the chemical nature of the amino acid side-chains affected. The changes can be easily accommodated in the rabbit muscle phosphorylase tertiary structure and appear to have little effect on the overall conformation. Interestingly the rat and human enzymes lack the carboxyl-terminal proline (residue 841) present in the rabbit enzyme and terminate at isoleucine (residue 840). The genetic basis for this difference in carboxyl termini is unknown. However, unlike the other amino acid changes, it cannot be accounted for by a single base-pair substitution. A comparison of the 3' untranslated regions in these cDNAs shows that there has been little constraint on the evolutionary divergence of most of this region (70% homology among the three species). There are, however, two repeated segments of DNA flanking the stop codons that are identical among all three species. Similar sequences are found within regions of DNA that contain a variety of transcriptional enhancers, suggesting the possibility that the repeats may be functional.
