With rapid progress being made in deciphering plant genomic sequences, determining the function of these genes is one of the main challenges that plant molecular biologists face today. Herbicidal inhibitors have been very useful for understanding gene function in at least two examples, represented by herbicidal inhibitors of hydroxyphenylpyruvate dioxygenase (HPPD) and deoxyxylulosephosphate reductoisomerase (DXR). In the first, an albino mutant of Arabidopsis isolated during the study of carotenoid biosynthesis was found to have an intact carotenoid biosynthetic pathway. A number of “bleaching herbicides” in development at about the same time (e.g., sulcotrione) produced similar symptoms by strongly inhibiting HPPD, a key enzyme in plastoquinone biosynthesis. Examination of the Arabidopsis mutant revealed that the HPPD gene had been inactivated in the albino plants. Inhibition of the HPPD pathway also led to reduced levels of tocopherol (vitamin E), an end product of the pathway. Further studies and manipulation of the pathway produced plants with significantly higher levels of vitamin E. This result is a clear demonstration of how an herbicidal inhibitor was able to lead to the identification of a gene that was responsible for a particular phenotype. As a second example, identification of fosmidomycin as a specific inhibitor of DXR in the recently elucidated nonmevalonate pathway of isopentenyl pyrophosphate (IPP) biosynthesis was instrumental in furthering the understanding of an important route to synthesis of many important terpenoid products.

In the δAsn20 Drosophila stock, the N-linked glycosylation site of opsin in Rl-6 receptors (Rhl) is absent. We used electroretinography (ERG), microspectrophotometry (MSP), and electron microscopy (EM) to quantify visual cell defects. Positive controls, w9, had wild type Rhl. MSP revealed minimal photopigment in δAsn20 for 6 days posteclosion; w9 had near normal visual pigment. ERG sensitivity and prolonged depolarizing afterpotential (PDA) were compared for δAsn20 and w9. δAsn20's Rl-6 function is decreased 100–fold at eclosion and diminishes until only R7/8 functions at 11 days. What little rhodopsin is routed to the rhabdomere functions. Morphometry showed smaller Rl-6 rhabdomeres in δAsn20 for 8 days posteclosion. Rhabdomeres in w9 were normal. A negative control, ninaE0117, a deletion of the Rhl gene, also has small rhabdomeres. δAsn20 and ninaE0117 lack the extreme rhabdomere elimination of ora (outer rhabdomeres absent), a nonsense mutant interrupting Rhl's coding sequence. δAsn20 and ora have surplus membrane while ninaE0117 does not. Freeze fracture reveals that δAsn20's rhabdomeric P-face particle count is as low as for vitamin A deprivation, consistent with an opsin defect. High particle density, organized into rows, is present in adjacent plasmalemma where surplus membrane accumulates. In summary, δAsn20 interferes with either synthesis, deployment, or maintenance of opsin.

Glycosylation variants of the virulent Leishmania major clone VI21 were generated by mutagenesis with N-methyl-N-nitroso-N-nitroguanidine and selected using the galactose-specific lectin Ricinus communis II (RCA II). Three mutants, 4B9, 1D1 and 1C12, which failed to bind RCA II, were found to have an altered expression of lipophosphoglycan (LPG), a molecule implicated in the attachment to host macrophages and survival within the phagolysosome. There were differences in the antigenicity, molecular weight and localization of LPG from mutant parasites as compared to V121. Expression of gp63, a surface molecule also implicated in attachment to macrophages, was unaltered. All 3 mutants caused disease when injected into genetically susceptible BALB/c mice but lesions developed at a much slower rate than those caused by the virulent V121 clone. This slow rate of lesion development did not correlate with promastigotes' ability to invade macrophages in vitro. Karyotype analysis showed that there was a reduction in the size of chromosome band number 2 in all 3 mutants. The differences in LPG and chromosome band 2 were retained by mutant clones following passage through mice, suggesting that these phenotypes are stable. Although the mutant parasites were infective and caused lesions, the changed structure of the LPG appeared to influence the virulence of the parasites.

Introduction. Black pepper (P. nigrum L.), the major world spice, is a climbing vine of historical, religious and economic importance. Landraces or farmer’s cultivars constitute the major component of this tropical vine's diversity. Normal spike formation. Berries of P. nigrum are produced in solitary, unbranched, axillary spikes. The apical bud of the plagiotropic branches transforms into an inflorescence (spike). Spike variant. Mutation in the floral meristem of black pepper could result in inflorescence proliferation. A variant with 100% of proliferating spikes was collected from a farmer’s plot, and then propagated by cuttings. The proliferating spikes are of indeterminate growth habit. Benefits. This natural mutant of Piper nigrum resulted in improved socioeconomic status of the farmer through sale of the rooted cuttings at a premium price. The variant can also be used as a donor for improvement of black pepper, besides as a novelty in potted pepper culture. Since berries of varying maturity are produced due to indeterminate growth, and since immature berries are rich in oleoresin, a single harvest will be yielding matured berries, which can be traded as black pepper of commerce, and half-matured berries suited to the value-added industry. Discussion. Mutations in the floral organ identity genes and their effect on altered flower/inflorescence development have been reported in the literature. In the case of spike proliferation, the transformation of the floral primordia in the mutant spike into inflorescence primordia has resulted in the modified spike architecture.

prp12-1 is one of the mutants defective in pre-mRNA splicing at a nonpermissive temperature in Schizosaccharomyces pombe. We found that the prp12+ gene encodes a protein highly homologous with a human splicing factor, SAP130/SF3b130, a subunit of a U2 snRNP-associated complex SF3b. Prp12p was shown to interact genetically with Prp10p that is a homolog of SAP155/SF3b155, another subunit in SF3b, suggesting that Prp12p is a functional homolog of human SAP130/SF3b130. Prp12p tagged with GFP is uniformly localized in the nuclear DNA region. In addition to pre-mRNA splicing defects, the prp12-1 mutant produced elongated cells, a typical phenotype of cell division cycle (cdc) mutants, suggesting a possible link between pre-mRNA splicing and cell-cycle progression. We examined kinetics of splicing defects in prp12-1 and several other prp mutants using northern blot hybridization and found that, among all the tested pre-mRNAs, only TfIId+ pre-mRNA with low splicing efficiency showed detectable splicing defects at the nonpermissive temperature in prp12-1. In addition, we found that other prp mutants with the cdc phenotype also showed differential splicing defects in tested pre-mRNAs at the nonpermissive temperature. On the other hand, prp mutants that do not exhibit the cdc phenotype showed a rapid and complete block of pre-mRNA splicing in all the tested pre-mRNAs at the nonpermissive temperature, indicating that prp mutants with weak splicing defects have a tendency to exhibit the cdc phenotype. These results suggest that the cdc phenotype in prp12-1 is caused by a selective reduction of spliced transcripts encoding a protein (or proteins) required for G2/M transition.

Tertiary folding of the 160-nt P4-P6 domain of the Tetrahymena group I intron RNA involves burying of substantial surface area, providing a model for the folding of other large RNA domains involved in catalysis. Stopped-flow fluorescence was used to monitor the Mg2+-induced tertiary folding of pyrene-labeled P4-P6. At 35 °C with [Mg2+] ≈ 10 mM, P4-P6 folds on the tens of milliseconds timescale with kobs = 15–31 s−1. From these values, an activation free energy ΔG[Dagger] of ∼8–16 kcal/mol is calculated, where the large range for ΔG[Dagger] arises from uncertainty in the pre-exponential factor relating kobs and ΔG[Dagger]. The folding rates of six mutant P4-P6 RNAs were measured and found to be similar to that of the wild-type RNA, in spite of significant thermodynamic destabilization or stabilization. The ratios of the kinetic and thermodynamic free energy changes Φ = ΔΔG[Dagger]/ΔΔG°′ are ≈0, implying a folding transition state in which most of the native-state tertiary contacts are not yet formed (an early folding transition state). The kobs depends on the Mg2+ concentration, and the initial slope of kobs versus [Mg2+] suggests that only ∼1 Mg2+ ion is bound in the rate-limiting folding step. This is consistent with an early folding transition state, because folded P4-P6 binds many Mg2+ ions. The observation of a substantial ΔG[Dagger] despite an early folding transition state suggests that a simple two-state folding diagram for Mg2+-induced P4-P6 folding is incomplete. Our kinetic data are some of the first to provide quantitative values for an activation barrier and location of a transition state for tertiary folding of an RNA domain.

The X-ray crystallographic structures of two mutants (K206Q and H207E) of the N-lobe of human transferrin (hTF/2N) have been determined to high resolution (1.8 and 2.0 Å, respectively). Both mutant proteins bind iron with greater affinity than native hTF/2N. The structures of the K206Q and H207E mutants show interactions (both H-bonding and electrostatic) that stabilize the interaction of Lys296 in the closed conformation, thereby stabilizing the iron bound forms.

Cell division was examined during leaf initiation in the slow-to-green mutant of Lolium temulentum L. to test the hypothesis that the cell cycle in the leaf primordium is a key regulator of the well characterized reduction in final leaf length in the mutant compared with that of the wild type. The cell doubling time (cdt, by colchicine method) was substantially longer in the youngest leaf primordium (YLP) of the mutant (107 h) than in the wild type (43 h) although the duration of the most rapid cell cycle (cc, by percentage labelled mitoses method) was between 18–20 h in each. As a consequence, the proportion of rapidly proliferating cells was only 20% in the mutant compared with 47% in the wild type. The size of the shoot apical meristem and the plastochron were similar between genotypes which indicates that the shoot meristem was largely buffered from the effects of the mutation. Mitotic cell area was also similar in the YLP of both genotypes. However, as the leaf elongated, mitotic cell area and interphase cell size were significantly larger in the mutant compared with the wild type. This change was coupled with a reduced number of cells per unit length of leaf in the mutant. The data are consistent in showing that the proportion of rapidly proliferating cells in the YLP (but not the rate of cell division) is a key parameter which influences growth of the leaf.