Clustering of hypervariable minisatellites in the proterminal regions of human autosomes

Six of the human minisatellites detected by DNA fingerprint probes have been localized by in situ hybridization to human metaphase chromosomes. These hypervariable loci are not dispersed at random in the human genome, but show preferential, though not exclusive, localization to terminal G-bands of human autosomes. Two of the proterminal minisatellites are very closely linked to other variable loci. Sequence analysis of one of these additional minisatellites suggests that the two linked minisatellites arose by independent amplification of different repeat units. The proterminal regions of human autosomes may therefore be rich in minisatellites, analogous to the pseudoautosomal terminal pairing region of human sex chromosomes that is similarly abundant in hypervariable minisatellites.     

The effects of low levels of chloramphenicol and methotrexate on somatic embryogenesis inCitrus

Summary The effects of the antibiotics methotrexate and chloramphenicol on somatic embryogenesis inCitrus were evaluated. Relatively low levels (0.1 to 1.0 μg/ml) of these antibiotics did not inhibit embryo production from undeveloped ovules of ‘Key’ lime [C. aurantifolia) (Christm.) Swing.]. Surprisingly, both antibiotics induced the formation of embryogenic callus in these cultures. This is usually a rare event in cultures of undevelopedCitrus ovules, and ‘Key’ lime is especially recalcitrant. The effects of these antibiotics on embryogenic callus appeared to be limited to the induction stage, because there was no consistent effect, either stimulatory or inhibitory, on established, lines of embryogenic callus. Florida Agricultural Experiment Station Journal Series No. 8958. This research was supported in part by a grant to Moore and Cline from the Competitive Grants Office of the SEA, USDA (85-CRCR-1-1623).     

Human recombinant insulin-like growth factor I. I. Development of a serum-free medium for clonal density assay of growth factors using balb/c 3T3 mouse embryo fibroblasts

Summary A serum-free clonal density growth assay was developed for the quantification of the biological activity of human recombinant insulin-like growth factor I (IGF-I). The assay measures IGF-I stimulated growth of Balb/c 3T3 cells cultured over 4 d on poly-d-lysine-coated plastic surfaces in a serum-free medium formulation composed of a 1∶1 (vol/vol) mixture of Ham's F12 and Dulbecco's modified Eagle's media, supplemented with 3.0 ng/ml bovine basic fibroblast growth factor (bFGF), 10 μg/ml human transferrin, 100 μg/ml ovalbumin, and 1.0 μM dexamethanose. Low-temperature trypsinization of serum-supplemented stock cultures combined with the use of poly-d-lysine-coated plates made it unnecessary to use serum or fibronectin to promote cell attachment and survival. Serum-free growth conditions were optimized with respect to the concentrations of the supplements. Addition of IGF-I resulted in 3.5-fold more cells than control cultures without IGF-I after 4 d. Deletion of bFGF resulted in no IGF-I stimulation of growth. The concentrations of various preparations of IGF-I required to achieve one-half maximal stimulation of cell number (ED₅₀), ranged between 1.25 and 4.7 ng/ml. In parallel assays, IGF-I was 6.6 times more potent than human recombinant insulin-like growth factor II and 32 times more potent than insulin. When cells were seeded into medium containing IGF-I, transferrin, ovalbumin, and dexamethasone but no bFGF, growth was minimal. Dose-response addition of bFGF showed an ED₅₀, of 0.9 ng/ml. The methods reported are useful to monitor the biological potency of recombinant and natural-source growth factors as well as providing a new means of studying the multiple growth factor requirements of Balb/c 3T3 cells in cultures. This work was supported by a contract from IMCERA Bioproducts, Inc.     

Human recombinant insulin-like growth factor I. II. Binding characterization and radioreceptor assay development using BALB/c 3T3 mouse embryo fibroblasts

Summary The binding of human recombinant insulin-like growth factor I (IGF-I) to BALB/c 3T3 mouse embryo fibroblasts has been characterized, resulting in the development of a radioreceptor assay. Binding of radioiodinated IGF-I (¹²⁵I-IGF-I) to washed monolayers of BALB/c 3T3 cells was specific, time dependent, and stable, being maximal after a 10-h incubation at 15°C with no loss of bound ligand or cells through 25 h. Scatchard analysis identified a class of high affinity binding sites with K _d =59.6 pM and an estimated 1.57×10⁵ receptors/cell. Half-maximal displacement of bound¹²⁵I-IGF-I occurred with 15 to 20 ng/ml unlabeled IGF-I competitor. Insulin-like growth factor II and insulin were far less effective competitors, providing halfmaximal displacement at concentrations of 130 to 170 ng/ml and 2 to 3 μg/ml, respectively. Epidermal growth factor, transforming growth factor type α, and acidic and basic fibroblast growth factors did not compete for¹²⁵I-IGF-I binding at 1 μg/ml. Cells fixed with glutaraldehyde before ligand binding did remain attached to culture dishes more tightly; however such pretreatment destroyed approximately 70% of ligand binding. Crosslinking data indicated that¹²⁵I-IGF-I binds specifically to a 330-kDalton receptor as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under nonreducing conditions. This receptor dissociated into 130-kDalton subunits when analyzed in the presence of dithiothreitol. This work was supported by a contract from IMCERA Bioproducts, Inc.     

The effect of pulse repetition rate on the delay sensitivity of neurons in the auditory cortex of the FM bat,Myotis lucifugus

1. Echo delay is the primary cue used by echolocating bats to determine target range. During target-directed flight, the repetition rate of pulse emission increases systematically as range decreases. Thus, we examined the delay tuning of 120 neurons in the auditory cortex of the bat, Myotis lucifugus, as repetition rate was varied. 2. Delay sensitivity was exhibited in 77% of the neurons over different ranges of pulse repetition rates (PRRs). Delay tuning typically narrowed and eventually disappeared at higher PRRs. 3. Two major types of delay-sensitive neurons were found: i) delay-tuned neurons (59%) had a single fixed best delay, while ii) tracking neurons (22%) changed their best delay with PRR. 4. PRRs from 1-100/s were represented by the population of delay-sensitive neurons, with the majority of neurons delay-sensitive at PRRs of at least 10-20/s. Thus, delay-dependent neurons in Myotis are most active during the search phase of echolocation. 5. Delay-sensitive neurons that also responded to single sounds were common. At PRRs where delay sensitivity was found, the responses to single sounds were reduced and the responses to pulse-echo pairs at particular delays were greater than the single-sound responses. In facilitated neurons (53%), the maximal delay-dependent response was always larger than the best single-sound responses, whereas in enhanced neurons (47%), these responses were comparable. The presence of neurons that respond maximally to single sounds at one PRR and to pulse-echo pairs with particular echo delays at other PRRs suggests that these neurons perform echo-ranging in conjunction with other biosonar functions during target pursuit.     

A molecular genetic linkage map of mouse chromosome 18 reveals extensive linkage conservation with human chromosomes 5 and 18

An interspecific backcross between C57BL/6J and Mus spretus was used to generate a molecular genetic linkage map of mouse chromosome 18 that includes 23 molecular markers and spans approximately 86% of the estimated length of the chromosome. The Apc, Camk2a, D18Fcr1, D18Fcr2, D18Leh1, D18Leh2, Dcc, Emb-rs3, Fgfa, Fim-2/Csfmr, Gnal, Grl-1, Grp, Hk-1rs1, Ii, Kns, Lmnb, Mbp, Mcc, Mtv-38, Palb, Pdgfrb, and Tpl-2 genes were mapped relative to each other in one interspecific backcross. A second interspecific backcross and a centromere-specific DNA satellite probe were used to determine the distance of the most proximal chromosome 18 marker to the centromere. The interspecific map extends the known regions of linkage homology between mouse chromosome 18 and human chromosomes 5 and 18 and identifies a new homology segment with human chromosome 10p. It also provides molecular access to many regions of mouse chromosome 18 for the first time.     

Glucocorticoid regulation of phenylethanolamine N-methyltransferase in vivo.

In vivo, supraphysiological doses of glucocorticoids are required to restore adrenal medullary phenylethanolamine N-methyltransferase (PNMT, E.C. 2.1.1.28) activity after hypophysectomy. However, in vitro, phenylethanolamine N-methyltransferase gene expression appears normally glucocorticoid-responsive. To explore this paradox, rats were given dexamethasone or the type II-specific glucocorticoid RU28362 (1-1000 micrograms/day), and adrenal phenylethanolamine N-methyltransferase activity and mRNA levels were determined. At low doses (1-30 micrograms/day), neither steroid altered mRNA whereas at higher doses (100-1000 micrograms/day), mRNA rose 10- to 20-fold, with dexamethasone approximately 3 times as potent as RU28362. In contrast, enzyme activity fell with low doses of either steroid, consistent with suppression of ACTH and endogenous steroidogenesis. At higher doses of RU28362, enzyme activity remained low and unchanged despite increased mRNA expression, whereas higher doses of dexamethasone progressively restored the enzyme to normal. These findings suggest 1) that glucocorticoid regulation of phenylethanolamine N-methyltransferase activity occurs largely independent of gene expression; 2) that glucocorticoid effects on enzyme activity are primarily indirect, probably through cosubstrate regulation and/or enzyme stabilization; and 3) that these effects are not mediated via a classical (type II) glucocorticoid receptor mechanism, given the high doses of dexamethasone and corticosterone required and the inability of RU28362 to mimic the effects of these less selective steroids.     

Behavioral response ofGraminella nigrifrons (Homoptera: Cicadellidae) to experimentally manipulated vibrational signals

Mate recognition for the leafhopper Graminella nigrifrons(Forbes) occurs when a male spontaneously emits a multisectional vibrational calling song to which females respond by emitting simple pulses. Significant differences were found among males in the duration, number of chirps, and chirp rate within sections of the song and the total song. Repeatability (proportion of total variation due to differences among males) of call features ranged from very low (0.04 for total chirps in song) to high (0.67 for section 3 chirp rate). However, song modification and playback experiments revealed that the variation in the measured song features was not important in determining whether a female will respond. Rather, female response depended only on the presence of two of the three types of pulses which comprise a chirp. These essential pulses were found within chirps of all call sections that contain chirps. Manipulation of chirp rates from 0.58 to 2.70 times the normal rate did not affect female response, nor did changing the period of silence between the essential pulse types from 0.25 to 1.75 times the normal period. These results suggest that components of the male calling song function in mate recognition but are not used by females to discriminate among conspecific males.