Photographic map of the polytene chromosomes of Cochliomyia hominivorax

Abstract.  Cochliomyia hominivorax (Coquerel) (Diptera: Calliphoridae) is one of the most important myiasis-causing flies and is responsible for severe economic losses to the livestock industry throughout the Neotropical region. A polytene chromosome map is an invaluable tool for the genetic analysis and manipulation of any species because it allows the integration of physical and genetic maps. Cochliomyia hominivorax has a diploid number of 12 chromosomes (2 n  = 12): five pairs of autosomes and one pair of sex chromosomes (XX/XY), which do not polytenize. We created a new photomap of the polytene chromosomes of C. hominivorax describing its five autosomes (chromosomes 2–6). Pupal trichogen cells, which have chromosomes with a high degree of polytenization, were used to elaborate this map. The photomap was made by comparing 20 different nuclei and choosing, for each chromosome segment, the region with the highest resolution. Thus, we present a new photomap of the five autosomes of this species, with a total resolution of 1450 bands.     

Analysis of nucleolus organizer regions (NORs) in mitotic and polytene chromosomes ofPhaseolus coccineus by silver staining and Giemsa C-banding

Chromosomes with active nucleolus organizer regions (NORs) were visualized in root tip metaphases ofPhaseolus coccineus using the silver staining technique. A mean number of 5.5 Ag-NORs per cell was observed in 54 cells from eight plants. In the endopolyploid nuclei of the suspensor the silver technique did not demonstrate the reported specificity for nucleolus organizer activity, because there was usually pale staining of nucleoli and preferential staining of heterochromatic regions in the polytene chromosomes including pericentromeric material, telomeres and NORs. The mean number of NORs per nucleolus as detected by this method was 5.8 (28 nucleoli analysed). Using a modified preparation technique, giant chromosomes stained pale, but nucleoli of suspensor cells displayed darkly silver staining internal domains, each of which originating from a nucleolus organizer.—Giemsa C-banding of endopolyploid suspensor nuclei revealed C-positive nucleolus organizers with darkly staining intranucleolar fibrils. The latter were frequently involved in inter-NOR associations. In 34 nucleoli analysed, the mean number of Giemsa C-positive NORs per nucleolus was 6.0.Dedicated to Professor Dr.Lothar Geitler on the occasion of his 80th birthday.     

Electron microscope analysis of the E polytene chromosome of Drosophila subobscura: region 70B to 72D

Revision of the reference map of the polytene chromosomes of Drosophila subobscura was started by means of the electron microscope. We present a map of regions 70B to 72D of the E chromosome obtained from squashed and thin-sectioned salivary glands. It was observed that the total number of bands in divisions 70B to 72D is considerably higher than those depicted in the reference map of Kunze-Mühl and Müller. Functional considerations are made of some regions that show puff structures.     

Effects of gamma-radiation on the organisation of polytene chromosomes ofGlyptotendipes salinus Michailova (Chironomidae,Diptera)

A series of laboratory experiments onGlyptotendipes salinus were carried out in order to assess cytogenetic effects of different doses of gamma-radiation on polytene chromosomes, isolated from salivary glands. Chrinonomid larvae (III–IV larval stage) were irradiated with doses varying from 0.05 to 1.00 Gy (5–100 rad) and were bred under laboratory conditions until the fourth larval stage. Cytogenetic slides were analyzed for an estimation of occurrence of changes in the organization of the polytene chromosomes caused by gamma-radiation. A specific heterochromatin effect was found in certain chromosomes of the investigated species after 1.00 Gy irradiation. Decondensation of the centromeric heterochromatin and increased functional activity of Balbiany ring 2 were observed in the fourth (G) chromosome. Regression of the nucleolus of the first (AB) chromosome was detected.     

Gibberellins in suspensor,embryo and integument from very young seeds of Phaseolus coccineus L.

Endogenous gibberellins (GAs) were extracted from suspensor, embryo and integument of very young seeds of Phaseolus coccineus L. and detected by combined gas chromatography-mass spectrometry (GC-MS). Results show the presence of one C₂₀-GA, GA₄₄ and five C₁₉-GAs in the suspensor: GA₁, GA₄, GA₅, GA₆ and GA₈, and four C₁₉-GAs in the integument: GA₁, GA₅, GA₆ and GA₈. Only traces of GA₁ and GA₅ were identified in the embryo. A compound structurally related to GAs was identified as tetrahydroxy-Kauranoic acid in suspensor, integument and, only in trace amounts, in the embryo.     

K+ Gradients in the Pulvinus of Phaseolus coccineus during Leaf Movement*

We used quantitative histochemistry to investigate the tissue-specific compartmentation of potassium in the laminar pulvinus of Phaseolus coccineus L. at day and night positions of diurnal leaf movement. The assay was based on the potassium-dependent activation of pyruvate kinase. Total potassium levels of pulvini were higher in the light than in the dark [0.88 and 0.57mol (kg dry weight)^?1, respectively]. Transverse compartmentation of potassium was studied on three tissue slices, representing the middle part, petiolar and laminar sides of individual pulvini. These were dissected further into 10 distinct subsamples (bundle; motor tissues: extensor, flexor; flanks). In the day position the amount of potassium in the extensor was higher than in the night position [1.92 and 1.50 mol (kg dry weight)^?1, respectively]. Flexor changes were opposite [1.13 and 1.65 mol (kg dry weight)^?1, respectively]. In the day position there was a steep and consistent increase in potassium content from the innermost to the outermost zones of the extensor. In the night position this was much more variable. Comparable gradients were not detected in flexor samples. Here highest amounts of potassium were recovered from the middle of the motor tissue. The data specify distinct tissue regions involved in osmotic adjustment during leaf movement in Phaseolus coccineus.     

Cytological localization of thePGIP genes in the embryo suspensor cells ofPhaseolus vulgavis L

Polygalacturonase-inhibiting protein (PGIP) is a cell wall protein which inhibits fungalendopolygalacturonases. A small gene family encodesPGIP in the genome of common bean, as indicated by Southernblot experiments performed at high-stringency conditions. Southern-blot analysis of DNA extracted from different cultivars ofPhaseolus vulgaris and fromPhaseolus coccineus showed length polymorphism of the hybridizing restriction fragments. The cytological localization of thePGIP genes was determined in polytene chromosomes of theP. vulgaris embryo suspensor cells. In-situ hybridization experiments using the clonedPGIP gene revealed labelling over a single region of the pericentromeric heterochromatin of chromosome pair X, next to the euchromatin, suggesting thatPGIP gene family may be clustered in one chromosomal region.     

On the Reversible Conjugation of [17-D2]GA20 in Seedlings of Phaseolus coccineus L.

After administering [17-D₂]GA₂₀ to Phaseolus coccineus L. cv. Preisgewinner seedlings, [17-D₂]GA₂₀-O-glucoside was identified by liquid chromatography (LC)/ESI-tandem mass spectrometry (MS). Likewise, by LC/ESI-tandem MS the metabolic formation of [17-D₂]GA₂₀ glucosyl ester was established. The application of both [17-D₂]-labeled GA₂₀ 13-O-glucoside and GA₂₀ glucosyl ester to Phaseolus coccineus L. seedlings resulted in free [17-D₂]GA₂₀ by gas chromatography/MS. The results demonstrate that conjugation of GA₂₀ and the reconversion of the glucosyl conjugates are concomitant processes in plants. Received October 27, 1998; accepted August 12, 1999     

A method for long-term micropropagation of Phaseolus coccineus L.

A method for long-term plant regeneration of Phaseolus coccineus L, is described. Shoot-tips and cotyledonary nodes cultured on a Murashige and Skoog medium supplemented with N⁶-benzylaminopurine, 10 M, and -naphthaleneacetic acid, 1M, formed multiple bud-shoots. These shoots were transferred to medium containing BAP 1 M, NAA 0.1 M, and gibberellic acid 3 M to promote shoot growth and further shoot multiplication. Rooting was achieved in medium with 11 M indole-3-acetic acid. Rooted plants grew to maturity and were fertile. Cultures have maintained their ability to regenerate plants for more than two years. A sample of 30 regenerated plants (R₀) was tested for chromosome number, all of them being diploid; seven isozymatic systems were electrophpretically analyzed in 82 R₀ regenerated plants. No differences were observed in their electrophoretic patterns in comparison with those shown by seedlings. Histological studies revealed the origin of buds from calluses via organogenesis.Abbreviations BAP N⁶-benzylaminopurine - 2,4-D 2,4-dichlorophenoxyacetic acid - GA₃ gibberellic acid - IAA indole-3-acetic acid - MS Murashige and Skoog (1962) medium - NAA -naphthaleneacetic acid - ADH alcohol dehydrogenase - GOT glutamic-oxaloacetic transaminase - MDH malate dehydrogenase - 6PGD 6-phosphogluconate dehydrogenase - PGI Phosphoglucose isomerase - PGM phosphoglucose mutase - SK shikimate dehydrogenase     

Laser‐assisted microdissection of polytene chromosomes from Diptera for the development of molecular markers

The structural details visible under the light microscope have made polytene chromosomes from Diptera much used in studies of the taxonomy, evolution and genetics of important biological models such as Drosophila and Chironomus, and the medical entomology of important disease vectors such as blackflies and mosquitoes. This paper describes the isolation of sections of polytene chromosomes from preserved wild‐caught blackflies using off‐the‐shelf laser microdissection microscopy and subsequent analysis of the DNA using polymerase chain reaction. This allows a direct link between the visible structure of the genome and the unknown DNA sequence, facilitating the development of molecular markers for population cytogenetics and cytotaxonomy.     

The transport and metabolism of gibberellins A1 and A5 in excised segments from internodes of Phaseolus coccineus

Jacobs, W. P., Beall, F. D. and Pharis, R. P. 1988. The transport and metabolism of gibberellins A₁ and A₅ in excised segments from internodes of Phaseolus coccineus. -Physiol. Plant. 72: 529–534. The transport and metabolism of gibberellins (GAs) ([³H]-GA, and [³H]-GA₅) of high specific radioactivity were investigated in excised segments from young internodes of Phaseolus coccineus L. Both GA₁ and GA₅ are native to this species and present in shoot tissue. The segments, 5.1 mm long, were incubated for 6 h in the horizontal position with agar donor blocks containing the [³H]-GA on the morphological apical or basal ends and with plain agar receiver blocks on the opposite end. At the end of incubation, the individual agar blocks were analyzed immediately for total radioactivity, or both blocks and intervening tissue were frozen and freeze-dried for later chromatographic analysis. The movement of both [³H]-GA, and [³H]-GA₅ was found to be consistently without polarity. However, approximately 5-fold more [³H]-GA, than [³H]-GA₅ was transported through the Phaseolus segments into receivers when equal amounts were in the donors. The extractable radioactivity from receiver blocks was primarily that of the donor GA. No putative GA conjugates were found in any class of receivers, but more GA metabolites were found in the free acid fraction from acropetal than basipetal receivers. Chromatographic analysis by reversed phase C₁₈ high performance liquid chromatography of the tissue segments showed that [³H]-GA, was metabolized more than [³H]-GA₅. Tissue adjacent to receiver blocks contained not only the precursor GA from the donor, but also polar ‘free GA metabolites’ and putative GA glucosyl conjugates. These results provide evidence that GA., which is the known ‘effector’ GA for elongation in shoot tissue of several species, is more effectively transported than GA₅ (a known precursor of GA₁) or than GA₁s more polar metabolites.     

Cellular double-stranded RNA in Phaseolus vulgaris

Summary High molecular weight double-stranded (ds) RNAs have been detected in apparently virus-free French (common) bean Phaseolus vulgaris cv. Black Turtle Soup (BTS). Several other bean cultivars were free of detectable high molecular weight dsRNAs. The dsRNAs have been partially characterized and have homology to the BTS genome as well as to the genomes of other bean cultivars. The T_m of hybrids formed between BTS DNA and denatured dsRNA have been estimated.     

Detection of rRNA and phaseolin genes on polytene chromosomes of Phaseolus coccineus by fluorescence in situ hybridization after pepsin pretreatment.

This is the first report of fluorescence in situ hybridization (FISH) on plant polytene chromosomes. Different protease pretreatments have been tested to improve fluorescence in situ hybridization FISH on polytene chromosomes of a plant, Phaseolus coccineus, with the aim to enable the detection of low-copy genes. The structural preservation of the chromosomes and the distinctness of the FISH signals were comparatively analysed with a probe for the ribosomal RNA genes after digestion with pepsin and trypsin. The pepsin pretreatment resulted in a general loosening of chromatin with good conservation of chromosome morphology and an increased number and density of signal points. The six nucleolus organizers exhibited significant differences in condensation. The pretreatment with pepsin enabled the detection of the low-copy genes encoding the seed storage protein phaseolin.     

Nurse cell polytene chromosomes of Drosophila melanogaster otu mutants: Morphological changes accompanying interallelic complementation and position effect variegation

Combinations of certain mutant alleles of the ovarian tumor gene permit the production of viable eggs. Two alleles that behave in this way are otu⁷ and otu¹. Females homozygous for either allele are sterile, and their ovarian nurse cells (NC) contain giant polytene chromosomes of various morphologies. Fertile flies (otu⁺ / otu⁺, otu⁻ / otu⁷, otu⁺ / otu¹¹) have endopolyploid nurse cells with typical dispersed chromosomes. Fertile hybrids (otu⁷ / otu¹¹) produce large numbers of polytene chromosomes comparable to, and often larger than, classic salivary gland (SG) chromosomes. Therefore, these otu hybrids provide a unique system for studying, at the chromosomal level, the activation and expression of genes functioning during oogenesis. The otu gene encodes a long and a short isoform. The normal long isoform appears to be responsible for the dispersion of chromosomes during the endomitotic DNA replications occurring in ovarian NCs. The genetic inactivation of euchromatic genes placed next to pericentric heterochromatin by a chromosomal rearrangement is accompanied by the compaction of corresponding chromosome regions. A comparative study of the manifestation of position-effect variegation for the polytene chromosomes of SG cells and NCs was made using the Dp(1;1)pn2b and Dp(1;f)1337 rearrangements. The percentage frequencies of block formation in the SG and NC nuclei for Dp (1;1) pn2b rearrangement were 92.6% vs. 15.8%, respectively; for Dp(1;f) 1337, these values were 56.8% vs. 9.7%. Therefore heterochromatin belonging to germ line chromosomes is in a configuration that is far less likely to inactivate inserted segments of euchromatin than is heterachromatin from somatic chromosomes. Dev. Genet. 20:163–174. 1997. © 1997 Wiley-Liss, Inc.     

Order of fertilization within the ovary in Phaseolus coccineus L. (Leguminosae)

Summary Phaseolus coccineus typically has six linearly arranged ovules per ovary. The three ovules near the stylar end of the fruit (positions one, two, and three) are more likely to produce mature seeds, to produce heavier seeds, and to produce more vigorous progeny than the ovules in positions near the peduncular/basal end of the fruit (ovule positions four, five, and six). We conducted a series of field experiments designed to supplement our understanding of the mechanisms determining these position effects. We found that approximately 98% of the ovules in 752 fruits were fertilized — about 0.6% of the stylar ovules were not fertilized, whereas 3.2% of the basal ovules were unfertilized. Moreover, we found that only about 49% of the ovules in these 752 fruits produced mature seeds. Over 60% of the stylar ovules produced mature seeds, whereas only 37% of the basal ovules produced mature seeds. Consequently, the proportion of fertilized ovules cannot explain the differences in seed maturation among the ovule positions. We found that after 6.5 h most of the fertilized ovules were located in the stylar ovule positions, and that there were no fertilized ovules in ovule positions five and six, indicating that the stylar ovules are fertilized first. When only the fastest growing pollen tubes were permitted to enter the ovary (due to exision of the style), only the ovules at the stylar end were fertilized, indicating that the ovule positions that are fertilized first are indeed fertilized by the fastest growing pollen tubes.On leave from the Escuela de Biologia, Universidad de Costa Rica, Cuidad Universitaria Rodrigo Facio, San Jose, Costa Rica, Central America