Dina Zevin-Sonkin

The correlation between the synthesis of skeletal muscle actin, myosin heavy chain, and myosin light chain and the accumulation of corresponding mRNA sequences during myogenesis ☆

Abstract Cloned cDNA probes were used to measure the accumulation of myosin heavy chain, myosin light chain 2, and actin mRNA during differentiation of rat skeletal muscle cell cultures. This was compared with the changes in the rate of synthesis of the corresponding proteins. Accumulation of those mRNA sequences was detectable a few hours before the onset of the phase of cell fusion; however, the main increase in hybridizable RNA occurred during the phase of rapid cell fusion. A close correlation was found between the amounts of mRNAs coding for these proteins and the rate of synthesis of the proteins. The results suggest that the activation of stored mRNA is not a major mechanism for controlling the time at which these proteins are synthesized.

Synthesis of plasma proteins in fetal, adult, and neoplastic human brain tissue☆

The synthesis of plasma proteins directed by mRNA from human brain tissues was studied by combining in vitro or in ovo translation of mRNAs with crossed immunoelectrophoresis of the mRNA-directed labeled polypeptides, followed by autoradiography of the washed plates. Poly(A)-containing mRNA was prepared from different developmental stages of fetal and postnatal human brain and also from primary glioblastomas and meningiomas. Several plasma protein-like polypeptides were identified in the autoradiographs by their migration coordinates in the two-dimensional gels, compared with immunoprecipitates formed by mature, unlabeled, stainable proteins. These included polypeptides migrating like Gc globulin, haptoglobin, fibrinogen, alpha-fetoprotein, transferrin, cholinesterase, and alpha 2-macroglobulin; other, yet unidentified plasma proteins, were also observed. In general, the synthesis of these plasma proteins appeared to be more pronounced in fetal and neoplastic brain tissues than in postnatal tissues. However, clear immunoprecipitates for some of these plasma proteins could also be detected in products directed by mRNA from particular regions of mature, normal brains, indicating that some synthesis of plasma proteins takes place in the human brain even as late as 40 years of age. mRNAs for several proteins were also identified in samples of neoplastic brain. mRNA for transferrin was identified in normal fetal and adult brain but not in either the glioblastomas or meningiomas studied. Microinjected Xenopus oocytes, in which post-translational processing occurs as well, were also used to translate fetal brain mRNA. Several plasma proteins could be detected in the translation products which were induced and stored in the oocytes. These included hemopexin, which could not be detected in the in vitro system. Others, such as cholinesterase, were found to be secreted by the oocytes. These findings indicate that different cell types in the human brain may produce and either store or secrete particular plasma proteins at defined stages in their development.

Accumulation of muscle-specific RNA sequences during myogenesis☆

Abstract DNA complementary to rat skeletal muscle polyadenylated RNA was enriched for sequences specific for terminal differentiation by hybridization to RNA extracted from cloned mononucleated myogenic cells and subsequent removal of the hybridized cDNA. The remaining cDNA (musclespecific cDNA) was hybridized to RNA extracted from primary skeletal muscle cultures harvested at short time intervals during differentiation. The experiments indicate that sequences specific for terminal differentiation accumulate close to the time of cell fusion, possibly a few hours prior to it. DNA complementary to polyadenylated muscle RNA was fractionated by hybridization to its template at a low R0t and separation of the hybridized (abundant) and nonhybridized (rare) cDNA. Hybridization of these fractions to RNA extracted from cultures harvested prior to or after cell fusion showed that the abundant cDNA is very much enriched for sequences specific for terminal differentiation.

The use of mRNA translation in vitro and in ovo followed by crossed immunoelectrophoretic autoradiography to study the biosynthesis of human cholinesterases.

Summary1.The synthesis of various cholinesterases in different fetal human tissues was studied usingin vitro andin ovo translation of poly(A)+ RNA, followed by crossed immunoelectrophoretic autoradiography.2.When unfractionated poly(A)+ mRNA from fetal brain, muscle, or liver was translatedin vitro, in the reticulocyte lysate cell-free system, polypeptides were synthesized which reacted with antibodies against either “true” acetylcholinesterase (acetylcholine hydrolase; EC 3.1.1.7) or “pseudo”, butyrylcholinesterase (acylcholine acylhydrolase; EC 3.1.1.8).3.The two nascent cholinesterases could be separated by crossed immunoelectrophoresis followed by autoradiography, suggesting that acetylcholinesterase and butytylcholinesterase are produced in all three tissues from nascent polypeptides containing different immunological domains.4.To examine whether the biosynthesis of cholinesterases includes posttranslational processing events,Xenopus oocytes were microinjected with mRNA from these tissues. Immunoelectrophoretic analysis of oocyte intracellular homogenates and incubation medium revealed various precipitation arcs, reflecting the synthesis and posttranslational processing of multiple forms of tissue-specific exported and intracellular acetylcholinesterase and butyrylcholinesterase.5.These findings demonstrate that polymorphic cholinesterases are produced from nascent polypeptide products which undergo further posttranslational processing events in a tissue-specific manner before they become mature compartmentalized cholinesterases.

Molecular Biology Approach to the Expression and Properties of Mammalian Cholinesterases

At the cholinergic synapse, the enzyme acetylcholinesterase terminates the electrophysiological response to the neurotransmitter acetylcholine (ACh) by degrading it very rapidly. Acetylcholine is a principal, and the most studied, neurotransmitter in all the higher eukaryotic organisms. The cholinergic acetylcholine receptor and the hydrolyzing enzyme, AChE, are thus the two major elements in a regulatory system that controls the response to ACh in numerous cell types, tissues, and organisms.

DENS (differential extension with nucleotide subsets): application to the sequencing of human genomic DNA and cDNA.

Here we describe further development of our method of DNA sequencing by Differential Extension with Nucleotide Subsets (DENS) and its application to the sequencing of human genomic DNA and full-insert cDNA. Essentially, DENS is primer walking without custom primer synthesis; instead, DENS uses a presynthesized library of octamer primers degenerate in two positions (4,096 tubes/sequences for a complete library). DENS converts an octamer selected from this library into a long primer on the template, at the intended site only. This is done using a two-step procedure which starts with a limited extension of the octamer (at 20 degrees C) in the presence of only two of the four possible dNTPs. The primer is extended by five bases or more at the intended priming site, which is deliberately selected to maximize the extension length (as are the two-dNTP set and the primer itself). The subsequent termination reaction at 60 degrees C then accepts the primer extended at the intended site, but not at alternative sites, where the initial extension (if any) is generally much shorter. This paper presents a set of rules for selection of DENS priming sites. We also compare different ways of template preparation for DENS sequencing. The data were obtained from primer walking on three human genomic DNA subclones of 3 to 4 kbp and four cDNA clones containing inserts of 1.9, 2.3, 3.8, and 4.9 kbp. Full-length sequences were obtained from both strands of each subclone by automated dye-terminator fluorescent DNA sequencing using DENS with degenerate octamer primers. We compared the following types of DNA templates: single-stranded and double-stranded phagemid DNA, double-stranded PCR products, asymmetric PCR products, and single-stranded DNA produced by digestion with Lambda Exonuclease of double-stranded PCR product phosphorylated at one end (Exo-PCR). While all of the preps were found to work, the best results were obtained with Exo-PCR and phagemid single-stranded DNA. Exo-PCR directly from overnight bacterial culture with no plasmid prep of any kind yielded templates for DENS as good as Exo-PCR from purified DNA. We found that the Tm of the differentially extended octamers is an important factor in the success of DENS. Clustering of successful reactions was clearly distinguished in the Tm range of 50-66 degrees C, with success rates of 70% for Exo-PCR and 65% for ss phagemid templates.