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P B Holm

Publications and source records attributed to P B Holm.

8 recordsLinked to original sources
PubMed2000

Transformation of barley by microinjection into isolated zygote protoplasts.

Barley zygote protoplasts were mechanically isolated, embedded in agarose droplets, and microinjected with a rice actin promoter Act1-gusA-nos gene construct. On average 62% of the cells survived the injection and of these 55% continued development into embryo-like structures and eventually to plants. PCR screening for the presence of a 307-bp fragment in the middle of the gusA gene showed that on average 21% of the derived structures contained this fragment. However, among the hundreds of injected zygotes, derived structures and regenerants we only found significant GUS expression in two cases (embryo-like structures nine days after injection). Two lines of green plants, derived from zygotes microinjected with linearized plasmid (line A147-1) or an isolated Act1-gusA-nos gene cassette (line A166-h) proved to be transgenic. Line A147-1 appeared to contain a single and intact copy of the expression cassette but a PCR based progeny analysis indicated the presence of additional shorter fragments of the cassette. Line A166-h appeared to contain a single fragment of the gusA gene that was transferred to the progeny as a single Mendelian trait. One additional fragment of the gusA gene was identified in this line. The present data show that transformation of barley by microinjection of DNA into isolated zygotes is feasible but also that gene expression rarely is achieved, possibly due to degradation of the introduced DNA.

Actins↗
PubMed1996

Engineering of the aspartate family biosynthetic pathway in barley (Hordeum vulgare L.) by transformation with heterologous genes encoding feed-back-insensitive aspartate kinase and dihydrodipicolinate synthase.

In prokaryotes and plants the synthesis of the essential amino acids lysine and threonine is predominantly regulated by feed-back inhibition of aspartate kinase (AK) and dihydrodipicolinate synthase (DHPS). In order to modify the flux through the aspartate family pathway in barley and enhance the accumulation of the corresponding amino acids, we have generated transgenic barley plants that constitutively express mutant Escherichia coli genes encoding lysine feed-back insensitive forms of AK and DHPS. As a result, leaves of primary transformants (T0) exhibited a 14-fold increase of free lysine and an 8-fold increase in free methionine. In mature seeds of the DHPS transgenics, there was a 2-fold increase in free lysine, arginine and asparagine and a 50% reduction in free proline, while no changes were observed in the seeds of the two AK transgenic lines analysed. When compared to that of control seeds, no differences were observed in the composition of total amino acids. The introduced genes were inherited in the T1 generation where enzymic activities revealed a 2.3-fold increase of AK activity and a 4.0-9.5-fold increase for DHPS. T1 seeds of DHPS transformants showed the same changes in free amino acids as observed in T0 seeds. It is concluded that the aspartate family pathway may be genetically engineered by the introduction of genes coding for feed-back-insensitive enzymes, preferentially giving elevated levels of lysine and methionine.

Amino Acids↗
PubMed1986

Ultrastructural analysis of meiotic recombination and chiasma formation.

Meiotic recombination appears to be mediated by recombination nodules, small electron dense spheres, associated with the central region of the synaptonemal complex. These structures are a prerequisite for, not the result of crossing over and they undergo complex structural, numerical and distributional changes before the final distribution of crossovers is achieved. In the organisms studied the nodules are placed randomly along and among the bivalents at zygotene. Subsequently, the distribution is modified to insure that each bivalent and generally each bivalent arm attain a nodule and that close spacing of nodules is minimized. Analysis of the number and location of recombination nodules at pachytene provides knowledge of the number and physical distribution of crossovers in the individual bivalents. Chiasmata originate from recombination nodules. At early diplotene the chiasma consists of a retained segment of synaptonemal complex containing a derivative of a recombination nodule. At late meiotic prophase the segment is eliminated after the formation of a chromatin chiasma.

Animals↗
PubMed1984

The synaptonemal complex, recombination nodules and chiasmata in human spermatocytes.

Reconstruction from serial sections of 120 human spermatocyte nuclei, ranging in stage from leptotene to anaphase I, has led to the following conclusions. The reconstructed nuclei form a continuous sequence of developmental stages. With few exceptions, all of the bivalents are identifiable by length, centromere index and morphological markers. Crossing over is accompanied by a transformation of the recombination nodule (RN) into a fusiform bar lying across the synaptonemal complex (SC). The evolution of the crossover distribution at late pachytene is regulated at two levels both involving the SC. Each bivalent displays a pattern of SC regions - domains - with high affinity for RNs in the middle and low affinity at the ends. The occurrence of crossing over in a domain reduces the probability of attachment of new RNs and causes RNs not yet involved in crossing over to be released from that domain. Positive interference between crossovers is a consequence of these phenomena. At diplotene the degradation of the SC is initiated in regions with low affinity for RNs and small segments of the SC are retained at the crossover sites until late diakinesis. The site of crossing over is only identifiable by an RN for a certain period of time during pachytene. The distribution of total crossovers in a nucleus is similar but not identical to the distribution of chiasmata as seen in the light microscope. The number of crossovers has been estimated to be 70 and exceeds the number of chiasmata determined by light microscopy by a factor of 1.4.

Animals↗
PubMed1982

Ultrastructural characterization of the meiotic prophase. A tool in the assessment of radiation damage in man.

The three-dimensional reconstruction of meiotic nuclei from serial sections micrographed in the electron microscope has provided information about man and several other organisms that is not obtainable by light microscopy or biochemical analysis. At zygotene, the previously unpaired chromosomes align and form synaptonemal complexes between homologous chromosome segments either by progressive initiation from the telomeres or by interstitial recognition. Chromosome and bivalent interlocking at zygotene is a regular phenomenon and occurs at a frequency of 0.7-4.0 per nucleus in samples of meiocytes analyzed from different organisms. This frequency is reduced to 0.1 per nucleus at pachytene. The interlockings are resolved by breakage and precise rejoining of the broken ends. This breakage and rejoining can also occur in the absence of the DNA nicking and repair involved in crossing-over. The synaptonemal complexes combining homologous chromosome segments are stabilized by recombination nodules, after which a second round of synaptonemal complex formation between as yet unpaired or unstably paired chromosome segments occurs, apparently for optimization of bivalent formation. Non-homologous pairing with the synaptonemal complex can take place in this phase of pachytene. Continuity between recombination nodules and chromatin chiasmata has been traced at the ultrastructural level but not all nodules lead to chiasmata. The distributions of recombination nodules among the bivalents and along the bivalents at successive stages of meiotic prophase show that the nodules are placed at random at early-zygotene after which bivalents without nodules have preference for the acquisition of these structures. Chiasma interference appears as a consequence of the limited number of recombination nodules available together with a decreased affinity of a bivalent arm with a nodule for additional ones. The relevance of these observations in the study of genetic damage by radiation is discussed.

Aneuploidy↗