RFLP mapping of the centromere of chromosome 4 in maize using isochromosomes for 4S.

The centromere of maize chromosome 4 was previously localized to a 26-cM interval using molecular markers and B-A translocations. The objective of the present study was to refine the placement of the centromere using secondary trisomics. Two independently isolated secondary trisomics (having an isochromosome plus two normal homologs) for 4S were recovered. RFLP analysis of populations segregating for them placed the centromere of chromosome 4 between bnl15.45 and bnl7.20, two RFLP loci that are 5.4-cM apart on the UMC map and 11.5-cM apart on the BNL map.

Analysis of total free and glucose-conjugated pyrethroid acid metabolites in tea infusions as hexafluoroisopropyl esters by gas chromatography with electron capture detection.

A gas chromatographic method with electron capture detection (GC-ECD) has been developed to quantitate the sum of free acids of pyrethroid metabolites, their glucosides, and their other base-cleavable conjugates that are extracted from tea leaves when a tea infusion is prepared. Four representative glucoside conjugates were synthesized; hydrolytic conditions were established; and extraction, derivatization and GC conditions were developed for analysis of these pyrethroid acid metabolites at > or = 0.01 ppm on dry tea. GC/mass spectrometry was used to confirm assignment of residues found in some tea samples.

Light and scanning electron microscopic observations of the canalicular system in human cellular cementum.

A scanning electron microscopic cast technique was used to determine the nature of the canalicular system in human cellular cementum. Prior light microscopic observations suggested the presence of two distinct types of lacunae: bone-like and a large irregular type generally confined to the interradicular region. Only the bone-like lacunae were visualized in the SEM cast preparations. The canalicular system associated with the bone-like lacunae was usually continuous from the surface of the dentin to the surface of the cementum in newly-erupted teeth. Casts having a sponge-like configuration were observed near the cementodentinal junction in some of the specimens from the interradicular region. The presence of these casts could not be predicted from prior light microscopic observations and it was concluded that they may represent infiltration of hypomineralized matrix rather than lacunae.

Analysis of Nondisjunction Induced by the R-X1 Deficiency during Microsporogenesis in Zea Mays L.

The r-X1 deficiency in maize induces nondisjunction at the second mitotic division during embryo sac formation. However, it was not known if this deficiency also induces nondisjunction during the microspore divisions. Microsporogenesis in plants lacking or containing this deficiency was compared using two approaches. First, chromosome numbers were determined in generative nuclei. Many (8.3%) of the generative nuclei in r-X1-containing plants were aneuploid; however, those from control plants were all haploid. Thus, this deficiency induces nondisjunction during the first microspore division. Second, nucleoli were analyzed in microspores. The only nucleolar organizing region in maize is on chromosome 6. If chromosome 6 underwent nondisjunction during the first microspore division, one nucleus in binucleate microspores would contain no nucleolus and the other would contain two nucleoli (or one nucleolus if the nucleoli fused). Only one (0.03%) microspore of this type was observed in control plants while 1.12% were found in r-X1-containing plants. Thus, the r-X1 deficiency induces nondisjunction of chromosome 6 during the first microspore division. However, both of the sperm nuclei in trinucleate microspores contained one nucleolus in r-X1-containing and control plants; thus, this deficiency does not induce nondisjunction of chromosome 6 (and presumably other chromosomes) during the second microspore division.

Use of monosomics to map cloned DNA fragments in maize.

Monosomic maize (Zea mays L.) plants were generated using the r-X1 deficiency system, and the monosomy was confirmed both genetically and cytologically. Genomic DNAs prepared from a group of plants, each monosomic for one chromosome, were digested with restriction enzymes, electrophoresed in agarose gels, and blotted onto nylon membranes. Hybridization of labeled cloned DNA fragments to these blots proved efficient in assigning each fragment to the chromosome from which it originated. Cloned DNA has previously contributed to loci detection through the use of the restriction fragment length polymorphisms (RFLPs), these loci subsequently being arranged into linkage groups by segregation analysis. In this study, these linkage groups were assigned to specific chromosomes, facilitating the construction of a linkage map for maize containing 112 RFLP loci. An additional 35 loci were also assigned to chromosomes by this method; however, the linkage relationships of these loci to other RFLP loci on each chromosome remains undetermined.

Scanning and transmission electron microscopy of tubular structures presumed to be human odontoblast processes.

Tubular structures interpreted as being odontoblast processes can be observed with the scanning electron microscope (SEM) on fractured dentin surfaces which have been demineralized and treated with collagenase. To confirm the nature of these structures, SEM preparations exhibiting similar tubular structures were subsequently examined with the transmission electron microscope (TEM). Newly-erupted human third molars were fractured buccolingually with heavy-gauge industrial nippers or sectioned mesiodistally with a Leitz saw microtome and fixed in glutaraldehyde. The exposed dentin surfaces were decalcified to a depth of approximately 500 microns and then treated with bacterial collagenase. Half of the specimens were critical-point-dried and coated for SEM. The other half were post-fixed and processed for TEM. After examination by SEM, the specimens were embedded and thin-sectioned for TEM. SEM observations of both the fractured and cut surfaces of dentin showed tubular structures running from the surface of the pulp to the dentino-enamel junction. When the SEM preparations were examined with TEM, the tubular structures were seen to be the inner sheath of the peritubular matrix, not odontoblast processes. In the specimens directly processed for TEM, the structures lying inside the sheath could be visualized clearly. In the outer two-thirds of the dentin, the tubules were essentially empty. Well-defined odontoblast processes were seen lying inside the sheath only in the inner dentin.

Canalicular communication in the cortices of human long bones.

A scanning electron microscopic (SEM) cast method that has been utilized to examine the internal structure of dentine was modified to examine canalicular communications in the cortices of a human femur and ulna. Although some preparations in which all of the matrix was removed were examined, etched preparations were found to be the most informative. Casts of lacunae and canaliculi along with the underlying matrix could be visualized in these preparations. In the femur, whose cortex exhibited a typical lamellar pattern, canalicular communication was seen between first and second generation osteons and occasionally between osteons and interstitial regions. The interstitial regions in the ulna appeared to be primarily woven rather than lamellar bone. Extensive communication between the outermost lacunae of osteons and interstitial regions was observed in the ulna.

Fibrin tissue adhesive and glass ceramic strut for ossicular chain replacement. Electrophysiologic and histologic findings.

The tolerance of the inner ear to fibrin tissue adhesive was tested. In experimental surgery on thirty-four chinchilla ears, Fibrin Sealant was applied to the inner ear contents by closing the oval window with a connective tissue graft dipped in the adhesive and gluing a Macor glass ceramic strut between this graft and tympanic membrane. No inner ear changes or tissue damage were found. The loss in auditory sensitivity monitored by auditory brain stem response thresholds was commensurate with the amount of loss to be expected with ossicular chain replacement. The findings suggest that this tissue glue can be safely applied to the labyrinth.

Scanning electron microscopic observations of casts of human dentinal tubules along the interface between primary and secondary dentine.

A scanning electron microscope cast technique was used to examine the interface between primary and secondary dentine in young and old human teeth. In addition, more traditional methods were used to examine this interface with light microscopy, identical regions being viewed before and after demineralization. No continuity was seen between the tubules in primary dentine and those in irregular secondary dentine in the scanning electron microscope preparations. These preparations did show the tubules to be continuous between primary dentine and regular secondary dentine in young and old teeth. Both the scanning electron microscopic and light microscopic observations suggested that regular secondary dentine becomes highly sclerosed in old teeth.

Relationship between Ureide N and N(2) Fixation, Aboveground N Accumulation, Acetylene Reduction, and Nodule Mass in Greenhouse and Field Studies with Glycine max L. (Merr).

The relationship between ureide N and N(2) fixation was evaluated in greenhouse-grown soybean (Glycine max L. Merr.) and lima bean (Phaseolus lunatus L.) and in field studies with soybean. In the greenhouse, plant N accumulation from N(2) fixation in soybean and lima bean correlated with ureide N. In soybean, N(2) fixation, ureide N, acetylene reduction, and nodule mass were correlated when N(2) fixation was inhibited by applying KNO(3) solutions to the plants. The ureide-N concentrations of different plant tissues and of total plant ureide N varied according to the effectiveness of the strain of Bradyrhizobium japonicum used to inoculate plants. The ureide-N concentrations in the different plant tissues correlated with N(2) fixation. Ureide N determinations in field studies with soybean correlated with N(2) fixation, aboveground N accumulation, nodule weight, and acetylene reduction. N(2) fixation was estimated by (15)N isotope dilution with nine and ten soybean genotypes in 1979 and 1980, respectively, at the V9, R2, and R5 growth stages. In 1981, we investigated the relationship between ureide N, aboveground N accumulation, acetylene reduction, and nodule mass using four soybean genotypes harvested at the V4, V6, R2, R4, R5, and R6 growth stages. Ureide N concentrations of young stem tissues or plants or aboveground ureide N content of the four soybean genotypes varied throughout growth correlating with acetylene reduction, nodule mass, and aboveground N accumulation. The ureide-N concentrations of young stem tissues or plants or aboveground ureide-N content in three soybean genotypes varied across inoculation treatments of 14 and 13 strains of Bradyrhizobium japonicum in 1981 and 1982, respectively, and correlated with nodule mass and acetylene reduction. In the greenhouse, results correlating nodule mass with N(2) fixation and ureide N across strains were variable. Acetylene reduction in soybean across host-strain combinations did not correlate with N(2) fixation and ureide N. N(2) fixation, ureide N, acetylene reduction, and nodule mass correlated across inoculation treatments with strains of Bradyrhizobium spp. varying in effectiveness on lima beans. Our data indicate that ureide-N determinations may be used as an additional method to acetylene reduction in studies of the physiology of N(2) fixation in soybean. Ureide-N measurements also may be useful to rank strains of B. japonicum for effectiveness of N(2) fixation.

Rhizobium japonicum Serogroup and Hydrogenase Phenotype Distribution in 12 States.

A survey was conducted in 1980 on 972 isolates of Rhizobium japonicum obtained from 65 soybean field locations in 12 states. Isolates were examined for the hydrogenase (Hup) phenotype and somatic serogroup identity. Only 20% of the isolates were Hup, with a majority of Hup isolates occurring in 10 of the 12 states. The most predominant serogroup was 31 (21.5%), followed by 123 (13.6%). Although most serogroups contained a majority of Hup isolates, marked differences occurred. None of the isolates in serogroup 135 were Hup, but 93% of the isolates in serogroup 122 were Hup. The serogroups with relatively high frequencies of Hup isolates (122 and 110) constitute only a small part (<5% each) of the R. japonicum field population in the 12 states.

Microradiographic and electron microscopic observations of the enamel-covered dentine in rat molars.

The enamel-covered primary and secondary coronal dentine in the molars of 90-day-old and 360-day-old rats was examined using microradiography. Some preparations were subsequently examined by transmission and scanning electron microscopy (SEM). Fractured dentine surfaces and methacrylate casts of the tubular system in the primary dentine were also examined with SEM. No microradiographic evidence of a hypermineralized peritubular matrix, such as that seen in man and other species, was seen in either young or old rats. Transmission and SEM confirmed the microradiographic findings. The tubule obliteration and extensive intra-luminal mineral deposits which have been reported in the enamel-free coronal dentine of the rat molar were not seen in the enamel-covered coronal dentine but some evidence of tubule obliteration was seen in the secondary dentine.

A different approach to immersion fixation of human dental pulp and odontoblast processes.

A low-speed diamond-edge rotary saw microtome was used to prepare freshly extracted human teeth for fixation. The teeth were sliced transversely 900 microns thick using saline as a coolant; the slices were immersed in glutaraldehyde fixative within 10 min of tooth extraction. After flat-embedding in Araldite, sections were reduced to 70 microns thickness and examined with light microscopy. Selected areas were processed for electron microscopy. In the light microscope, the entire pulp appeared to be well fixed. Electron microscopy showed that the contents of most of the tubules near the predentine were well fixed and microfilaments and microtubules were present in odontoblast processes. The quality of fixation with this method was at least as good as other methods applicable to teeth with the advantage that the integrity of the entire dental pulp was preserved.

An improved technique for producing casts of the internal structure of hard tissues, including some observations on human dentine.

A method for producing plastic casts of the internal structure of dentine has been described by Marten, Hallsworth and Buckley, J. Microsc. 112, 345-352 (1978). The main drawback of the method is that the casts collapse when they are air-dried. In the present investigation a freeze-drying technique was used to eliminate this problem. In addition, subtle variations in methodology were necessary to produce optimal casts of different hard tissues. These are described in detail and initial observations of the internal structure of human coronal and radicular dentine are also included. Special attention was given to the structure of Tomes' granular layer.

A Comparative Study of the Physiology of Symbioses Formed by Rhizobium japonicum with Glycine max, Vigna unguiculata, and Macroptilium atropurpurem.

Although Rhizobium japonicum nodulates Vigna unguiculata and Macroptilium atropurpurem, little is known about the physiology of these symbioses. In this study, strains of R. japonicum of varying effectiveness on soybean were examined. The nonhomologous hosts were nodulated by all the strains tested, but effectiveness was not related to that of the homologous host. On siratro, compared to soybean, many strains reversed their relative effectiveness ranking. Both siratro and cowpea produced more dry matter with standard cowpea rhizobia CB756 and 176A22 than with the strains of R. japonicum. Strains USDA33 and USDA74 were more effective with siratro and cowpea than with soybean. The strain USDA122 expressed high rates of hydrogenase activity in symbiosis with the cowpea as well as the soybean host. The strains USDA61 and USDA74 expressed low levels of hydrogenase activity in symbiosis with cowpea, but no activity was found with soybean. Our results indicate host influence for the expression of hydrogenase activity, and suggest the possibility of host influence of nitrogenase for the allocation of electrons to N(2) and H(+).

A grinding method for producing sections of large areas of plastic embedded tissues.

We have developed a method utilizing relatively thick ground sections of plastic embedded tissue which affords the resolution obtained with 0.5 micrometers cut sections. The sections, which are permanently affixed to plastic microscope slides, are much larger in area than ultramicrotome sections. Additional advantages are: sections can be destained and restained and selected areas can be examined with various forms of electron microscopy. Autoradiographic studies are also possible. Although the method has a broader application, it is particularly useful in examining the interface between hard and soft tissues.

Fast-growing rhizobia isolated from root nodules of soybean.

Fast-growing rhizobia have been isolated from soybean root nodules collected in China. These new isolates are physiologically distinct from slow-growing soybean rhizobia. They formed effective nitrogen-fixing associations with wild soybean and an unbred soybean cultivar from China, but were largely ineffective as nitrogen-fixing symbionts with common commercial cultivars of soybeans.

Maize pollen test systems to detect nondisjunction.

Three pollen test systems to detect the induction of nondisjunction in maize are actively being explored. (1) Each member of a tetrad of haploid microspores produced by meiosis contains a single chromosome 6 which carries the only nucleolar organizing region in the maize genome. Thus, each member of a normal tetrad contains one nucleolus. If nondisjunction took place at the first or second meiotic division, 2:2:0:0 or 2:1:1:0 tetrads would be produced respectively. (2) If a male parent carrying a dominant endosperm marker is crossed by a female carrying a recessive allele of this gene, all normal kernels would be heterozygous and would express the dominant phenotype in their endosperm. If nondisjunction of the chromosome carrying this gene took place at the second microspore division and the nullisomic and disomic sperm fertilized the polar nuclei and egg of a given embryo sac respectively, an exceptional kernel is produced which would express the recessive endosperm phenotype and contain a trisomic embryo. (3) Complementing null mutations of genes expressed in individual pollen grains can be utilized to detect nondisjunction. Normal haploid pollen grains from plants heterozygous for two complementing null alleles of a locus would each express the recessive phenotype. If nondisjunction took place during either meiotic division, disomic pollen grains containing both alleles could be produced expressing the dominant phenotype due to complementation. We are exploring this test system utilizing appropriate alcohol dehydrogenase mutants.