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The introduction of an inverted repeat to the 5′ untranslated leader sequence of a transgene strongly inhibits gene expression (2000)

Groenewald, J.-H. ; Hiten, N. F. ; Botha, F. C.

Springer

Plant cell reports 19 (2000), S. 1098-1101

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ISSN: 1432-203X

Keywords: Key words Transgene expression ; Stem-loop structure ; Inverted repeat ; 5′ Untranslated region ; Plant transformation ; AbbreviationsCaMV: Cauliflower mosaic virus ; GUS:β-Glucuronidase ; MCS: Multiple cloning site ; MS: Murashige and Skoog (l962) ; Ubi-1: Maize polyubiquitin ; UTR: Untranslated region

Source: Springer Online Journal Archives 1860-2000

Topics: Biology

Notes: Abstract Stable secondary structures in naturally occurring 5′ untranslated regions have been shown to down-regulate both transcription and translation. We introduced a synthetic GC-rich inverted repeat to the leader sequence of a transgene to determine its influence on gene expression. The addition of a 54-bp inverted repeat led to a more than 90% reduction in transient gene expression, while the addition of an inverted repeat of 42-bp reduced gene expression by 88%. Complete removal of the inverted repeat abolished this inhibiting effect. This dramatic decrease in transgene expression is probably due to the formation of stable stem-loop structures in the 5′ untranslated region of the reporter gene. The secondary structure energy of the putative stem-loop structures in the 54-bp and 42-bp repeats are –64.6 and –40.3 kcal mol–1, respectively. In comparison, the most stable stem-loop structure in the control construct's leader has a free energy of –15.4 kcal mol–1. This has important implications for the design of expression vectors where the recombination of multiple cloning sites and other 5′ leader sequences can lead to the introduction of inverted repeats that has the potential of forming stable stem-loop structures and resulting in a significant decrease in gene expression.

Type of Medium: Electronic Resource

URL: http://dx.doi.org/10.1007/s002990000262

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Use of PCR-based methodologies for the determination of DNA diversity between Saccharum varieties (1996)

Harvey, M. ; Botha, F. C.

Springer

Euphytica 89 (1996), S. 257-265

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ISSN: 1573-5060

Keywords: DNA diversity ; microsatellites ; PCR ; RAPDs ; Saccharum ; telomeres

Source: Springer Online Journal Archives 1860-2000

Topics: Agriculture, Forestry, Horticulture, Fishery, Domestic Science, Nutrition

Notes: Summary In this study, two PCR-based methodologies were evaluated for potential use in the determination of DNA diversity between 20 commercial sugarcane hybrids and 6 ‘outgroup’ varieties of S. spontaneum, S. officinarum and hybrids from early in the genealogy. The first method involved PCR amplification of sugarcane DNA in the presence of random, decamer primers (RAPDs), while the second protocol utilized specific microsatellite and telomere sequences as primers. A total of 41 RAPD primers (356 loci) were screened across the varieties of which 15 (160 loci) were used in the calculation of DNA diversity (expressed% similarity). This varied from 61 to 95%, with most of the commercial varieties showing more than 80% similarity in their DNA. The RAPD data indicated that there had been a gradual decline in DNA diversity (84% reduction) from the early inter-specific crosses to the commercial hybrids, probably as a result of backcrossing and in-breeding strategies used in the previous 5 to 6 generations of sugarcane breeding. The microsatellite and telomere data produced a much greater range in DNA similarity values (25–91%), probably due to the fact that these primers detect highly variable regions of the genome. It is suggested that these specific primers would not be suitable for determination of DNA diversity, but could be used more effectively in the development of a methodology for routine, rapid identification of sugarcane varieties.

Type of Medium: Electronic Resource

URL: http://dx.doi.org/10.1007/BF00034614

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Stability and potential use of RAPD markers in a sugarcane genealogy (1995)

Huckett, B. I. ; Botha, F. C.

Springer

Euphytica 86 (1995), S. 117-125

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ISSN: 1573-5060

Keywords: DNA ; genealogy ; PCR ; polymorphisms ; RAPDs ; Saccharum ; sugarcane

Source: Springer Online Journal Archives 1860-2000

Topics: Agriculture, Forestry, Horticulture, Fishery, Domestic Science, Nutrition

Notes: Summary A complete ancestral history of the recently developed and closely related South African commercial sugarcane varieties N11 and NCo376, which differ markedly in their response to sugarcane mosaic virus (SCMV), was elucidated from archival records. The genealogy spans seven generations, starting with early intraspecific crosses between varieties of Saccharum officinarum and interspecific crosses between S. officinarum and either S. spontaneum or S. barberi. In total, the genealogy comprises 38 different varieties. Genomic DNA samples from N11 and NCo376 respectively were screened for polymorphisms using the PCR-RAPD technique. Ten polymorphic fragments ranging in molecular size from 317 to 1263bp were identified from a total of 1159 loci amplified with 100 random decamer primers. Two of the 10 polymorphic fragments were shown to be consistently present in N11 (resistant) and absent in NCo376 (susceptible), while 8 showed the reverse occurrence. The primers producing the polymorphisms were used to screen genomic DNA samples from all 19 varieties representing the genealogy. Results have indicated that (1) specific PCR-RAPD generated polymorphic fragments can indeed be identified across the seven generations; (2) certain fragments are sufficiently definitive to be used as markers to trace parentage; (3) the validity of documented crosses and/or the authenticity of germplasm material may be questioned using this technique, and (4) there is the potential to subject the markers to linkage analysis once a full and accurate assessment of the SCMV resistance phenotype is obtained.

Type of Medium: Electronic Resource

URL: http://dx.doi.org/10.1007/BF00022017

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Respiratory metabolism and gene expression during seed germination (1992)

Botha, F. C. ; Potgieter, G. P. ; Botha, A. -M.

Springer

Plant growth regulation 11 (1992), S. 211-224

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ISSN: 1573-5087

Keywords: Germination ; energy metabolism ; gene expression ; regulation ; respiration

Source: Springer Online Journal Archives 1860-2000

Topics: Agriculture, Forestry, Horticulture, Fishery, Domestic Science, Nutrition

Notes: Abstract Oxygen uptake and carbon dioxide release rapidly increase in seeds during imbibition. The oxygen uptake is associated with oxidative phosphorylation through cytochrome oxidase. During the early stage of germination substrate level phosphorylation may also contribute to ATP production. All indications suggest that this route of ATP production is insignificant during aerobic germination. However, during oxygen stress, substrate level phosphorylation does significantly contribute to ATP production in some species. Carbohydrate oxidation plays a significant role in the germination process. Up to two thirds of the carbon from carbohydrate breakdown enters the tricarboxylic acid cycle through the phosphoenolpyruvate carboxylase reaction. This anapleurotic input into the Krebs cycle most probably reflects the high demand on intermediates from the cycle for biosynthesis. The extent to which other substrates are utilized for respiration is uncertain. Information regarding the levels of key metabolites and enzymes, as well as their cellular distribution is limited. The involvement of gene expression in the regulation of respiratory metabolism is poorly characterised. Several genes which have been cloned are only expressed during germination. With the exception of the early methionine labeled polypeptide, little is known about the function of these genes.

Type of Medium: Electronic Resource

URL: http://dx.doi.org/10.1007/BF00024560

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