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T D McKnight

Publications and source records attributed to T D McKnight.

8 recordsLinked to original sources
PubMed1995

Fluorescent in situ hybridization of a bacterial artificial chromosome.

Fluorescent in situ hybridization (FISH) of a 130 kilobase cotton (Gossypium hirsuitum L.) bacterial artificial chromosome (BAC) clone containing a high proportion of single-copy DNA produced a large pair of FISH signals on the distal end of the long arm of a pair of chromosomes of the D-genome species G. raimondii Ulbr. and produced a fainter pair of signals on a small submetacentric pair of chromosomes of the A-genome species G. herbaceum L. The signals were synthetic with a nucleolar organizer region in G. raimondii and G. herbaceum. Signal pairs were easily recognized in interphase and metaphase cells either with or without suppression of repetitive sequences with unlabeled G. hirsutum C0t-1 DNA. High quality FISH results were consistently obtained and image analysis was not required for viewing or photography. Results indicate that FISH of BAC clones is an excellent tool for the establishment of new molecular cytogenetic markers in plants and will likely prove instrumental in the development of useful physical maps for many economically important crop species.

Bacteria↗
PubMed1994

Sites of accumulation of the antitumor alkaloid camptothecin in Camptotheca acuminata.

Camptothecin is an anticancer and anti-viral alkaloid produced by the Chinese tree Camptotheca acuminata (Nyssaceae). Despite previous reports of low levels of anticancer activity in leaves of Camptotheca acuminata, we have discovered that camptothecin accumulates to approximately 0.4% of the dry weight of young leaves. This level is 1.5-fold higher than that of the seeds and 2.5-fold higher than that of the bark, the two currently used sources of the drug. As the leaves mature, the concentration and absolute amount of camptothecin decreases rapidly. The high levels of camptothecin in young leaves could provide an easily harvested, non-destructive source of this important drug.

Camptothecin↗
PubMed1993

Expression of a 3-hydroxy-3-methylglutaryl coenzyme A reductase gene from Camptotheca acuminata is differentially regulated by wounding and methyl jasmonate.

We have isolated a gene, hmg1, for 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR) from Camptotheca acuminata, a Chinese tree that produces the anti-cancer monoterpenoid indole alkaloid camptothecin (CPT). HMGR supplies mevalonate for the synthesis of the terpenoid component of CPT as well as for the formation of many other primary and secondary metabolites. In Camptotheca, hmg1 transcripts were detected only in young seedlings and not in vegetative organs of older plants. Regulation of the hmg1 promoter was studied in transgenic tobacco using three translational fusions (-1678, -1107, -165) with the beta-glucuronidase (GUS) reporter gene. Histochemical analysis of plants containing each of the three promoter fusions showed similar developmental and spatial expression patterns. In vegetative tissues, GUS staining was localized to the epidermis of young leaves and stems, particularly in glandular trichomes. Roots showed intense staining in the cortical tissues in the elongation zone and light staining in the cortex of mature roots. hmg1::GUS expression was also observed in sepals, petals, pistils, and stamens of developing flowers, with darkest staining in the ovary wall, ovules, stigmas, and pollen. Leaf discs from plants containing each of the translational fusions showed a 15- to 20-fold wound induction of hmg1::GUS expression over 72 h; however, this increase in GUS activity was completely suppressed by treatment with methyl jasmonate. Taken together, these data show that a 165-bp fragment of Camptotheca hmg1 promoter is sufficient to confer developmental regulation as well as wound induction and methyl jasmonate suppression of GUS expression in transgenic tobacco.

Acetates↗
PubMed1989

In situ hybridization of biotinylated DNA probes to cotton meiotic chromosomes.

A modified procedure for in situ hybridization of biotinylated probes to meiotic chromosomes of cotton has been developed with high retention of squashed cells on slides, preservation of acid-fixed chromosome morphology, exceptionally low levels of background precipitate at nonspecific hybridization sites and improved photomicrographic recording. Salient features of the techniques include pretreatment of slides before squashing, cold storage of squash preparations, and use of interference filters for distinguishing precipitate from chromatin. A cloned 18S/28S ribosomal DNA fragment from soybean was biotinylated via nick-translation and hybridized to microsporocyte meiotic chromosomes of cotton (Gossypium hirsutum L. and G. hirsutum L. X G. barbadense L.). Enzymatically formed precipitate from streptavidin-bound peroxidase marked the in situ hybridization. In situ hybridization of biotinylated probes to cotton meiotic chromosomes adds the specificity and resoltion of in situ hybridization to the chromosomal and genomic perspectives provided by meiotic cytogenetic analyses. Molecular cytogenetic analyses of meiotic cells offer certain inherent analytical advantages over analyses of somatic cells, e.g., in terms of mapping, and for studying fundamental biological and genetic problems, particularly for organisms that are not amenable to somatic karyotypic analysis.

Biotin↗
PubMed1986

Nucleotide sequence and molecular evolution of two tomato genes encoding the small subunit of ribulose-1,5-bisphosphate carboxylase.

We have isolated and sequenced two cDNA clones (LESS5 and LESS17) encoding the small subunit of ribulose-1,5-bisphosphate carboxylase of tomato (Lycopersicon esculentum). At the nucleotide level, the protein-coding regions of these genes are 85% conserved, while the untranslated 3' regions are only 55% conserved. Comparison with rbcS genes from other species of Solanaceae suggests that the tomato LESS5 gene, the Nicotiana tabacum NTSS23 gene and the Petunia hybrida SSU8 gene are orthologous members of the rbcS gene family. In addition, the tomato gene LESS17, and the Petunia hybrida gene SSU611, may also be orthologous, since their untranslated 3' regions are related. There is a large difference between the two tomato rbcS genes in the frequency of the CG dinucleotide. This difference may reflect different levels of methylation, and therefore expression, of the tomato genes. Many of the differences involving the CG dinucleotide can be represented as transitions between C and T on the noncoding strand. Such changes are consistent with observations that methylated cytosines are hot-spots for transitions.

Base Sequence↗
PubMed1984

A simplified and efficient vector-primer cDNA cloning system.

A simplified, efficient, and versatile vector-primer cDNA cloning system is presented. The dimer-primer system is a modification of the method of Okayama and Berg (1982) with the following features: (i) the vector-primer molecules are more rapidly and reliably prepared by virtue of the elimination of an endonuclease digestion and the agarose gel purification step from the original method, and (ii) the final cDNA products contain polylinkers at both cDNA-vector junctions, simplifying the size analysis, subcloning, and sequencing of inserts. The system is highly efficient, yielding greater than 10(5) transformants using 1 microgram mRNA and 1 pmol of vector-primer ends, with 75% or more of the transformants having inserts. The ability of the system to produce clones of full-length or near full-length is demonstrated by the analysis of 32 ribulose-1,5-bisphosphate carboxylase small subunit cDNA clones from tomato.

Animals↗
PubMed1983

Characterization and preliminary mapping of cauliflower mosaic virus transcripts.

Using Northern blot analysis we have studied the transcription of the CM4-184 Ga. strain of cauliflower mosaic virus (CaMV) DNA. This analysis reveals that this CaMV strain, like the Cabb-BS and the Cabb B-JI strains, produces both a genomic length transcript and a 1900-nucleotide (nt) transcript during infection. In addition, we detect an 1800-nt PA+ transcript mapping primarily to the EcoRI-c region of the virus, and three apparent minor viral-specific PA + RNAs of 4900, 4500, and 4300 nt. We also report the presence of two small viral single-stranded DNAs produced during infection, and show that both DNAs are derived from the EcoRI-b region of the virus.

Chromosome Mapping↗