p53 is frequently mutated in Burkitt''s lymphoma cell lines.

A panel of 12 Burkitt's lymphoma cell lines and four other B cell lines were tested for the presence of mutations in p53. Protein analysis using a mutant-specific antibody and sequencing of both cDNA and genomic DNA revealed changes relative to the standard p53 protein sequence in 12 of the 16 lines studied, including 10 of the BL lines. Mutation of p53 in the BL lines was usually accompanied by loss of the other allele of p53. Testing of the mutated p53 cDNAs for gain of transforming activity or loss of growth suppression activity showed that several of the BL mutants were functionally altered from wild-type p53.     

Postnatal expression of glutamate decarboxylases in developing rat cerebellum

The recent identification of two genes encoding distinct forms of the GABA synthetic enzyme, glutamate decarboxylase (GAD), raises the possibility that varying expression of the two genes may contribute to the regulation of GABA production in individual neurons. We investigated the postnatal development the two forms of GAD in the rat cerebellum. The mRNA for GAD₆₇, the form which is less dependent on the presence of the cofactor, pyridoxal phosphate (PLP), is present at birth in presumptive Purkinje cells and increases during postnatal development. GAD₆₇ mRNA predominates in the cerebellum. The mRNA for GAD₆₅, which displays marked PLP-dependence for enzyme activity, cannot be detected in cerebellar cortex by in situ hybridization until P7 in Purkinje cells, and later in other GABA neurons. In deep cerebellar nuclei, which mature prenatally, both forms of GAD mRNA can be detected at birth. The amounts of immunoreactice GAD and GAD enzyme activity parallel changes in mRNA levels. We suggest that the delayed appearance of GAD₆₅ is coincident with synapse formation between GABA neurons and their targets during the second postnatal week. GAD₆₇ mRNA may be present prior to synaptogenesis to produce GABA for trophic and metabolic functions.Special issue dedicated to Dr. Eugene Roberts.     

Experimental documentation of natural hybridization inCactaceae: Origin of Lloyd's Hedgehog Cactus,Echinocereus ×lloydii

The origin ofEchinocereus ×lloydii Britt. & Rose, pro sp. (Lloyd's Hedgehog Cactus) was investigated using comparative morphology, cytology, biochemistry, and particularly, artificial hybridization. Numerous artificial crosses between the putative parentsE. coccineus Engelm. (a species of claret-up cactus) andE. dasyacanthus Engelm. (Texas Rainbow Cactus) were successful, resulting in the production of hundreds of seeds with hybrid embryos. The F₁ hybrid progeny (i.e., syntheticE. ×lloydii) grew to sexual maturity in about four and one-half years, whereupon successful backcrosses and F₂ generation hybrids were also obtained. The known F₁ hybrids closely approximated naturalE. ×lloydii. The fertility of these syntheticE. ×lloydii was high, like their natural counterparts. The populations ofE. ×lloydii in Pecos County, Texas are inferred to have originated as the result of natural interspecific hybridization. It is assumed thatE. ×lloydii or similar plants may arise wherever the parental taxa grow sympatrically.     

Effect of monensin on receptor recycling during continuous endocytosis of asialoorosomucoid

The binding of asialoglycoproteins to their liver cell receptor results in internalization of the ligand-receptor complex. These complexes rapidly appear in intracellular compartments termed endosomes whose acidification results in ligand-receptor dissociation. Ligand and receptor subsequently segregate: ligand is transported to lysosomes and is degraded while receptor recycles to the cell surface. The proton ionophore monensin prevents acidification of endosomes and reversibly inhibits this acid-dependent dissociation of ligand from receptor. The present study determined the effect of monensin treatment of short-term cultured rat hepatocytes on cell-surface-receptor content, determined both by their binding activity and immunologically, following continuous endocytosis of asialoorosomucoid. Inclusion of 5 microM monensin in the incubation medium reduced the number of immunologically detectable cell-surface receptors by 20% in the absence of ligand. During continuous endocytosis of asialoorosomucoid, inclusion of monensin resulted in a 30-40% reduction of cell-surface receptor detectable either by ligand binding or immunologically. These results suggest that the reduced liver-cell-surface content of receptor in monensin is due to intracellular trapping of ligand-receptor complexes. The reduction of surface receptor during monensin incubation in the absence of ligand suggests that "constitutive recycling" of plasma membrane components also requires intracellular acidification.     

Roles of H1 domains in determining higher order chromatin structure and H1 location

Peptides derived from calf thymus H1 and rat liver H1, comprising only the globular and COOH-terminal domains of the intact molecule and therefore lacking NH2-terminal domains, have been shown by reconstitution to be as effective as the complete H1 molecule in inducing higher-order-chromatin structure. As the globular domain of H1 alone cannot induce chromatin folding, our results demonstrate that this function is primarily controlled by the COOH-terminal domain of the molecule. Surprisingly, these peptides do not locate correctly with respect to the nucleosome. This is demonstrated by their failure to confer upon reconstitutes the ability to protect DNA fragments of chromatosome length when digested with micrococcal nuclease. The precise placement of the H1 molecule (globular domain) with respect to the nucleosome is shown to be influenced by the "tail" domains of both H1 and the core histones.     

Contrasting effects of Escherichia coli single-stranded DNA binding protein on synthesis by T7 DNA polymerase and Escherichia coli DNA polymerase I (large fragment). Evidence that binding protein inhibits trans-lesion synthesis by polymerase I

The effect of Escherichia coli single-stranded DNA binding protein (SSB) on DNA synthesis by T7 DNA polymerase and E. coli DNA polymerase I (large fragment) using native or aminofluorene-modified M13 templates was evaluated by in vitro DNA synthesis assays and polyacrylamide gel electrophoresis analysis. The two polymerase enzymes displayed differential responses to the addition of SSB. T7 DNA polymerase, a enzyme required for the replication of the T7 chromosome, was stimulated by the addition of SSB whether native or modified templates were used. On the other hand, E. coli DNA polymerase I was slightly stimulated by the addition of SSB to the native template but substantially inhibited on modified templates. This result suggests that DNA polymerase I may be able to synthesize past an aminofluorene adduct but that the presence of SSB inhibited this trans-lesion synthesis. Polyacrylamide gels of the products of DNA synthesis by polymerase I supported this inference since SSB caused a substantial increase in the accumulation of shorter DNA chains induced by blockage at the aminofluorene adduct sites.     

Transition from somatic to meiotic pairing and progressional changes of the synaptonemal complex in spermatocytes of Aedes aegypti

Aedes aegypti spermatocytes were reconstructed from electron micrographs. The species has tight somatic pairing of the chromosomes, and there are therefore no classical leptotene and zygotene stages, but rather a gradual transition from somatic pairing to meiotic pairing (= pachytene). The term prepachytene has been used for the transitory stage. The first visible sign of impending meiosis was a reorganization of the chromatin, which resulted in the formation of spaces (synaptic spaces) in the chromatin, about the width of the synaptonemal complexes (SCs). Diffuse material, possibly precursor material for the SC, was present in the spaces. Later short pieces of complex were formed throughout the nucleus. Late prepachytene, pachytene, and diplotene complexes were reconstructed. Each chromosome occupied a separate region of the nucleus. The complexes became progressively shorter from prepachytene (maximum complement length 289 m) to diplotene (175 m). The thickness of the SCs increased from prepachytene to pachytene and probably decreased again during diplotene. At the beginning of diplotene the lateral elements (LEs) separated, and the single LEs became two to three times thicker than the LEs of the SC. The centromeres were at all stages attached to the nuclear membrane, whereas the telomeres were free in the nucleoplasm during pachytene and diplotene. A heterochromatic marker was present on chromosome 1 near the sex determining locus, and a diffuse marker on chromosome 3 near the nucleolus organizer region. After breakdown of the complexes, polycomplexes were present in the nucleus.     

Seasonal Bacterial Production in a Dimictic Lake as Measured by Increases in Cell Numbers and Thymidine Incorporation

Rates of primary and bacterial production in Little Crooked Lake were calculated from the rates of incorporation of H¹⁴CO₃⁻ and [methyl-³H]thymidine, respectively. Growth rates of bacteria in diluted natural samples were determined for epilimnetic and metalimnetic bacterial populations during the summers of 1982 and 1983. Exponential growth was observed in these diluted samples, with increases in cell numbers of 30 to 250%. No lag was observed in bacterial growth in 14 of 16 experiments. Correlation of bacterial growth rates to corresponding rates of thymidine incorporation by natural samples produced a conversion factor of 2.2 × 10¹⁸ cells produced per mole of thymidine incorporated. The mass of the average bacterial cell in the lake was 1.40 × 10⁻¹⁴ ± 0.05 × 10⁻¹⁴ g of C cell⁻¹. Doubling times of natural bacteria calculated from thymidine incorporation rates and in situ cell numbers ranged from 0.35 to 12.00 days (median, 1.50 days). Bacterial production amounted to 66.7 g of C m⁻² from April through September, accounting for 29.4% of total (primary plus bacterial) production during this period. The vertical and seasonal distribution of bacterial production in Little Crooked Lake was strongly influenced by the distribution of primary production. From April through September 1983, the depth of maximum bacterial production rates in the water column was related to the depth of high rates of primary production. On a seasonal basis, primary production increased steadily from May through September, and bacterial production increased from May through August and then decreased in September.     

SERGE, the subcellular site of initial hepatic glycogen deposition in the rat: a radioautographic and cytochemical study

Hormonal control of hepatic glycogen and blood glucose levels is one of the major homeostatic mechanisms in mammals: glycogen is synthesized when portal glucose concentration is sufficiently elevated and degraded when glucose levels are low. We have studied initial events of hepatic glycogen synthesis by injecting the synthetic glucocorticoid dexamethasone (DEX) into adrenalectomized rats fasted overnight. Hepatic glycogen levels are very low in adrenalectomized rats, and DEX causes rapid deposition of the complex carbohydrate. Investigation of the process of glycogen deposition was performed by light and electron microscopic (EM) radioautography using [3H]galactose as a glycogen precursor. Rats injected with DEX for 2-3 h and [3H]galactose one hour before being killed displayed an increasing number of intensely labeled hepatocytes. EM radioautography revealed silver grains over small (+/- 1 micron) ovoid or round areas of the cytosome that were rich in smooth endoplasmic reticulum (SER) and contained a high concentration of small dense particles. These distinct areas or foci of SER and presumptive glycogen (SERGE) were most numerous during initial periods of glycogen synthesis. After longer exposure to DEX (4-5 h) more typical deposits of cytoplasmic glycogen were evident in the SERGE regions. Several criteria indicated that the SERGE foci contained glycogen or presumptive glycogen: resemblance of the largest dense particles to beta-glycogen particles in EM; association of 3H-carbohydrate with the foci; removal of particles and label with alpha-amylase; and positive reaction with periodic acid-chromic acid-silver methenamine. The concentration of SER in the small foci and the association of newly formed glycogen particles with elements of SER suggest a role for this organelle in the initial synthesis of glycogen.