Biomedical subjects
Publications and source records attributed to A R Means.
A 15-kilobase pair EcoRI chick DNA fragment, containing both the termination codon UGA and the 5'-portion of the structural ovomucoid gene, has been cloned in lambda phage Charon 4A by in vitro packaging. Restriction mapping and electron microscopic analyses of this cloned DNA have revealed that the structural ovomucoid gene sequences are separated by at least six intervening sequences.
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Two proteins in the rat, androgen binding protein (ABP) and the cytoplasmic receptor (CR), have high affinity and limited capacity for binding androgens. To determine the structural requirements for binding with high affinity, each protein was partially purified and the ability of over 100 steroids to compete with [3H]dihydrotestosterone (17 beta-hydroxy-5 alpha-androstan-3-one) for binding sites was assessed. The results indicate marked differences in the steroid specificities of the two proteins. Some alterations of dihydrotestosterone at C-2 or C-2 and C-3 increase binding to ABP two to four-fold. Similarly, the affinity of 17 beta-hydroxy-7 alpha-methyl-4-estren-3-one for ABP increases two-fold when a double bond is created at C-14. Addition of a methyl group in the alpha position at C-7 or C-17, or an ethinyl group at C-17 cause little change in affinity; however, modifications at C-11 and C-17 beta, and deletion of the methyl group at C-10 significantly impair binding to ABP. Binding to the CR is maintained or increased by deletion of the methyl group at C-10. Binding is lessened by modifications at C-3 and C-17 beta. Most alterations at C-2, C-7, C-11, and C-17 alpha have only minor effects on binding to the CR. These studies should provide a molecular basis for predicting the effects of specific structural modifications. When some modifications at C-2 or C-2 and C-3 are combined with changes at C-17 beta, the resulting steroids retain very high affinity for ABP and very limited binding to the CR. Such steroids may provide a means for assessing the function of ABP.
Five independent clones containing the natural chicken ovomucoid gene have been isolated from a chicken gene library. One of these clones, CL21, contains the complete ovomucoid gene and includes more than 3 kb of DNA sequences flanking both termini of the gene. Restriction endonuclease mapping, electron microscopy and direct DNA sequencing analyses of this clone have revealed that the ovomucoid gene is 5.6 kb long and codes for a messenger RNA of 821 nucleotides. The structural gene sequence coding Ifor the mature messenger RNA is split into at least eight segments by a minimum of seven intervening sequences of various sizes. The shortest structural gene segment is only 20 nucleotides long. All seven intervening sequences are located within the peptide coding region of the gene, and the sequences at the 5' and 3' untranslated regions of the mRNA are not interrupted by intervening sequences. The DNA sequences of the regions flanking the 5' and 3' termini of the gene have been determined. Thirty nucleotides before the start of the messenger RNA coding sequence is the heptanucleotide TATATAT, which is also present in a similar location relative to the chicken ovalbumin gene and other unique sequence eucaryotic genes. This sequence resembles that of the Pribnow box in procaryotic genes where a promoter function has been implicated. Seven nucleotides past the 3' end of the gene is the tetranucleotide TTGT, a sequence found to be present at identical locations as either TTTT or TTGT in other eucaryotic genes that have been sequenced. These conserved DNA sequences flanking eucaryotic genes may serve some regulator function in the expression of these genes.
Indirect immunofluorescence was used to determine the distribution of calmodulin in the mitotic apparatus of rat kangaroo PtK2 and Chinese hamster ovary (CHO) cells. The distribution of calmodulin in PtK2 cells was compared to the distribution of tubulin, also as revealed by indirect immunofluorescence. During mitosis, calmodulin was found to be a dynamic component of the mitotic apparatus. Calmodulin first appeared in association with the forming mitotic apparatus during midprophase. In metaphase and anaphase, calmodulin was found between the spindle poles and the chromosomes. While tubulin was found in the interzonal region throughout anaphase, calmodulin appeared in the interzone region only at late anaphase. The interzonal calmodulin of late anaphase condensed during telophase into two small regions, one on each side of the midbody. Calmodulin was not detected in the cleavage furrow. In view of the differences in the localization of calmodulin, tubulin, and actin in the mitotic apparatus, experiments were designed to determine the effects of various antimitotic drugs on calmodulin localization. Cytochalasin B, an inhibitor of actin microfilaments, had no apparent effect on calmodulin or tubulin localization in the mitotic apparatus of CHO cells. Microtubule inhibitors, such as colcemid and N2O, altered the appearance of tubulin- and calmodulin-specific fluorescence in mitotic CHO cells. Cold temperature (0 degrees C) altered tubulin-specific fluorescence of metaphase PtK2 cells but did not alter calmodulin-specific fluorescence. From these studies, it is concluded that calmodulin is more closely associated with the kinetichore-to-pole microtubules than other components of the mitotic apparatus.
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A Ca2+-dependent regulator protein of cyclic 3':5'-nucleotide phosphodiesterase (EC 3.1.4.17) has previously been isolated from rat testis and shown to be a heat-stable, Ca2+-binding protein with a molecular weight of approximately 17,000. The Ca2+-dependent regulator protein is also structurally similar to troponin-C, the Ca2+-binding component of muscle troponin and Ca2+ mediator of muscle contraction. The present report describes a partial amino acid sequence of the Ca2+-dependent regulator. The protein (148 amino acids) is 50% homologous with skeletal muscle troponin-C, but is 11 residues shorter than the muscle protein. The Ca2+-dependent regulator protein has an NH2-terminal sequence of acetyl-Ala-Asp-Glu, a COOH-terminal sequence of Thr-Ala-Lys and 1 residue of epsilon-trimethyllysine located at position 115. All of these properties are distinct from those of other homologous Ca2+-binding proteins. These properties may account for the biological specificities demonstrated by these proteins as compared to the Ca2+-dependent regulator protein. Based on the sequence and a comparison of the Ca2+-dependent regulator protein to other calcium-binding proteins, our data support the view that all of these moecules contain common sequences, especially at their proposed metal-binding sites.
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Calcium-dependent regulator protein is a low molecular weight (17,000), thermostable, calcium binding protein which is structurally homologous to skeletal muscle troponin C. This protein is present in all nonmuscle cells and has been shown to decorate stress fibers in interphase cells by indirect immunofluorescence. Using this procedure we have investigated the distribution of the protein during mitosis of eukaryotic cells. As the cells enter prophase, the distinct cytoplasmic localization disappears commensurate with the dissolution of the cytoskeleton. The regulator protein seems to be randomly distributed throughout the prophase cell, including the region around the condensed chromosomes. However, at prometaphase, it is localized in association with the half-spindles of the mitotic apparatus. Through metaphase and most of anaphase, the protein remains localized between the chromosomes and the poles of the spindle. During late anaphase the protein is also found in the interzone region but rapidly condenses into two small regions, one on each side of the midbody that separates the daughter cells. The regulator protein is not localized in the cleavage furrow during telophase, whereas actin is demonstrable in this region. Indeed, placement of the protein during mitosis is distinct from both that of actin and that of tubulin. The localization of calcium-dependent regulator protein during mitosis suggests that it may mediate the calcium effects on the mitotic apparatus and thus play a role in chromosome movement.
The Ca2+-dependent regulator (CDR) protein of cyclic nucleotide phosphodiesterase is a low molecular weight, acidic, Ca2+-binding protein which has been implicated in a number of Ca2+-dependent enzymatic functions. Indirect immunofluorescence has revealed that CDR is specifically associated with the chromosome-to-pole region of the mitotic apparatus during metaphase-anaphase in a pattern distinctly different from that of similar cultured cells stained with antitubulin. This characteristic localization in the mitotic half-spindle suggested a role for CDR in the control of microtubule assembly-disassembly during mitosis. Thus, CDR was examined for its effects on microtubule polymerization in vitro. It was determined that stoichiometric concentrations of CDR and a homologous Ca2+-binding protein, skeletal muscle troponin C, both inhibited and reversed microtubule assembly in a Ca2+-dependent manner. CDR-dependent inhibition of in vitro microtubule assembly occurred at physiological Ca2+ concentrations (approximately 10 micron) that, in the absence of CDR, caused only a slight reduction in polymerization. At Ca2+ concentrations in the low physiological range (less than 1 micron), no inhibition was observed. These biochemical results, together with the immunofluorescent localization of CDR in the mitotic half-spindle, provide evidence that Ca2+ is an endogenous regulator of microtubule disassembly through the activity of CDR.
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The hormonal and testicular effects of hemicastration have been examined using rodent models. When rats were hemicastrated at 5 days of age, significant testicular hypertrophy was noted within 5 days. Hypertrophy decreased as the age at hemicastration approached 20 days and did not occur in rats 45 days of age or older. The changes in testicular weight were associated with significant (P less than 0.001) increases in serum FSH values from 10-20 days, but no significant alterations in the intratesticular concentration of testosterone occurred. Hemicastration also caused significant hypertrophy in testes depleted of germ cells. This was reflected by a substantial increase in testicular protein and DNA when compared to intact controls at 10, 15, and 20 days of age; however, the concentration of protein/microgram DNA was increased only at 15 days. These data indicate that 1) changes in serum FSH and not intratesticular testosterone are associated with the testicular hypertrophy which follows unilateral castration of immature animals; 2, a significant proportion of the hypertrophy can be attributed to non-germinal cells, the Sertoli cell being the prime candidate;3) the increase in testicular weight is primarily the result of an increase in cell number.
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