A highly sensitive northern blot assay detects multiple proenkephalin a-like mRNAs in human caudate nucleus and pheochromocytoma

Total RNA from post mortem human caudate nucleus, cerebellum, cerebral cortex and pheochromocytoma tissues has been prepared. Northern blot analysis, using a single-stranded human proenkephalin A antisense probe (cRNA), revealed the existence of two different proenkephalin A-like sequences in the human caudate nucleus and pheochromocytoma RNA extracts of approximately 1400 and 1000 nucleotides in length respectively, whereas no specific RNA bands could be detected in the cortex and only the 1400 nucleotide band was present in the cerebellum. Under highly stringent hybridization conditions, the proenkephalin A-like RNA bands still appear, indicating that the detected RNA species have either identical or a closely related sequence to that of the wellcharacterized human proenkephalin A mRNA sequence.     

Gene expression during CAM induction under salt stress in Mesembryanthemum: cDNA library and increased levels of mRNA for phosphoenolpyruvate carboxylase and pyruvate orthosphosphate dikinase

Mesembryanthemum crystallinum responds to high salinity in the soil by shifting the mode of carbon assimilation from the C3 mode to Crassulacean acid metabolism (CAM). Several enzymes of carbon metabolism have increased apparent activities in the CAM mode, including phosphoenolpyruvate carboxylase (PEPcase) and pyruvate orthophosphate dikinase (PPDK). We have identified cDNA clones for PEPcase and PPDK by immunological screening of a cDNA library constructed in the protein expression vector lambda gt11. The clones were characterized by immunoblotting and RNA blotting techniques. RNA blotting showed that during CAM induction the steady-state level of mRNAs for both PEP case and PPDK increased.Abbreviations IPTG isopropyl thiogalactoside - PEP phosphoenolpyruvate - PEPcase phosphoenolpyruvate carboxylase - PPDK pyruvate orthophosphate dikinase - Xgal-5 bromo-4-chloro-3-indolyl-beta-D-galactopyranoside     

Phylogenetic interrelationships of the barbets (Aves: Capitonidae) and toucans (Aves: Ramphastidae) based on morphology with comparisons to DNA-DNA hybridization

A phylogenetic analysis of the interrelationships of the barbets (Capitonidae) and the toucans (Aves: Ramphastidae, Superfamily Ramphastoidea) is presented. Thirty-two morphological characters from the literature and independent osteological observations were analysed. Character polarity was determined by outgroup comparison to the Picidae, Indicatoridae, Galbulidae, Bucconidae and Coraciiformes. Four alternative phylogenetic hypotheses were compared: (1) the overall most parsimonious morphological phylogeny, (2) the most parsimonious morphological phylogeny in which the capitonids and ramphastids were hypothesized as monophyletic sister groups, and (3) and (4) the most parsimonious hypotheses for the evolution of the morphological characters within two proposed DNA-DNA hybridization phylogenies of the ramphastoids. The analysis focused on the higher level relationships of ramphastids and capitonids and interrelationships among capitonid genera. Two cladistic analyses were performed using 26 phylogenetically informative characters, and the PAUP and CONTREE computer alogorithms. The most parsimonious morphological phylogeny required fewer character changes and had a lower consistency index than any of the alternative hypotheses but congruence between the most parsimonious phylogeny and the second, revised DNA-DNA hybridization hypothesis was very high. Based on these results the monophyly of the Capitonidae is rejected. The ramphastids and the Neotropical capitonids form a well corroborated clade within the pantropical ramphastoid radiation. Neither the African, Asian nor New World capitonids is monophyletic. The genus Trachyphonus is the sister group to all other capitonids and ramphastids. The sister group to the ramphastids is the genus Semnornis. The interrelationships of the Old World capitonids excluding Trachyphonus are not completely resolved by these morphological data but one of the alternative phylogenetic resolutions is presented as a preliminary hypothesis. The clades in this resolved phylogeny are diagnosed and the palaeontology and biogeography of the ramphastoids arc-reviewed in light of this new evidence. A phylogenetic classification is proposed in which the Capitonidae is rejected and the capitonids and ramphastids are placed in seven subfamilies of the Ramphastidae.     

A molecular and cytogenetic survey of major repeated DNA sequences in tomato (Lycopersicon esculentum)

Summary The major families of repeated DNA sequences in the genome of tomato (Lycopersicon esculentum) were isolated from a sheared DNA library. One thousand clones, representing one million base pairs, or 0.15% of the genome, were surveyed for repeated DNA sequences by hybridization to total nuclear DNA. Four major repeat classes were identified and characterized with respect to copy number, chromosomal localization by in situ hybridization, and evolution in the family Solanaceae. The most highly repeated sequence, with approximately 77000 copies, consists of a 162 bp tandemly repeated satellite DNA. This repeat is clustered at or near the telomeres of most chromosomes and also at the centromeres and interstitial sites of a few chromosomes. Another family of tandemly repeated sequences consists of the genes coding for the 45 S ribosomal RNA. The 9.1 kb repeating unit in L. esculentum was estimated to be present in approximately 2300 copies. The single locus, previously mapped using restriction fragment length polymorphisms, was shown by in situ hybridization as a very intense signal at the end of chromosome 2. The third family of repeated sequences was interspersed throughout nearly all chromosomes with an average of 133 kb between elements. The total copy number in the genome is approximately 4200. The fourth class consists of another interspersed repeat showing clustering at or near the centromeres in several chromosomes. This repeat had a copy number of approximately 2100. Sequences homologous to the 45 S ribosomal DNA showed cross-hybridization to DNA from all solanaceous species examined including potato, Datura, Petunia, tobacco and pepper. In contrast, with the exception of one class of interspersed repeats which is present in potato, all other repetitive sequences appear to be limited to the crossing-range of tomato. These results, along with those from a companion paper (Zamir and Tanksley 1988), indicate that tomato possesses few highly repetitive DNA sequences and those that do exist are evolving at a rate higher than most other genomic sequences.     

Proteinase inhibitors I and II in fruit of wild tomato species: Transient components of a mechanism for defense and seed dispersal

The juice of unripe fruit from a wild species of tomato, Lycopersicon peruvianum (L.) Mill., LA 107, contains over 50% of its soluble proteins as the sum of two proteinase inhibitors. These are the highest levels of proteinase inhibitors and highest percentage of soluble proteins as proteinase inhibitors of any plant or animal tissue found to date. Fruit of the modern tomato, L. esculentum Mill., contains only negligible quantities of the two inhibitors. The two proteinase inhibitors in the fruit of L. peruvianum are members of the Inhibitor I and II families previously found in potato tubers and in leaves of wounded potato and tomato plants. The levels of the two inhibitors in the unripe fruit decrease significantly during ripening. Unripe fruit from other wild Lycopersicon species such as L. parviflorum Rick, Kesicki, Fobes et Holle, L. hirsutum Humb. et Bonpe., L. pimpinellifolium Mill., and other lines of L. peruvianum contain moderate levels of the inhibitors that also decrease during ripening. Another wild tomato species, L. pennellii Corr., is similar to L. esculentum in not containing the two proteinase inhibitors in either unripe or ripe fruit. The transient levels of the inhibitors in fruit of wild species indicate that they are present in unripe fruit as defensive chemicals against insects, birds or small mammals and their disappearance during ripening may render them edible to facilitate seed dispersal. High levels of mRNAs coding for Inhibitors I and II in unripe fruit of L. peruvianum, LA 107, indicate that strong promoters may regulate the developmentally expressed proteinase-inhibitor genes in tomato fruit that may have a substantial potential for use in genetic-engineering experiments to enhance the production of large quantities of proteinase inhibitors or other proteins in field tomatoes.Abbreviations poly(A)⁺ mRNA polyadenylated mRNA - SDS-PAGE sodium dodecyl sulfate-polyacrylamide electrophoresis Project 1791, College of Agriculture and Home Economics Research Center, Washington, State University     

Acquisition of embryogenic potential in carrot cell-suspension cultures

Embryogenic suspension cultures of domesticated carrot (Daucus carota L.) are characterized by the presence of proembryogenic masses (PEMs) from which somatic embryos develop under conditions of low cell density in the absence of phytohormones. A culture system, referred to as starting cultures, was developed that allowed analysis of the emergence of PEMs in newly initiated hypocotyl-derived suspension cultures. Embryogenic potential, reflected by the number of FEMs present, slowly increased in starting cultures over a period of six weeks. Addition of excreted, high-molecular-weight, heat-labile cell factors from an established embryogenic culture considerably accelerated the acquisition of embryogenic potential in starting cultures. Analysis of [³⁵S]methionine-labeled proteins excreted into the medium revealed distinct changes concomitant with the acquisition of embryogenic potential in these cultures. Analysis of the pattern of gene expression by in-vitro translation of total cellular mRNA from starting cultures with different embryogenic potential and subsequent separation of the [³⁵S]methionine-labeled products by two-dimensional polyacrylamide gel electrophoresis demonstrated a small number of abundant in-vitro-translation products to be present in somatic embryos and in embryogenic cells but absent in nonembryogenic cells. Several other in-vitro-translation products were present in explants, non-embryogenic and embryogenic cells but were absent in somatic embryos. Hybridization of an embryoregulated complementary-DNA sequence, Dc3, to RNA extracted from starting cultures showed that the corresponding gene is expressed in somatic embryos and PEMs but not in non-embryogenic cells.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - cDNA complementary DNA - PAGE polyacrylamide gel electrophoresis - PEM proembryogenic mass     

以点杂交方法对人β型血珠蛋白基因的染色质结构与功能进行的研究

我采用点杂交的方法,对人β型血珠蛋白基因簇的染色质结构与基因转录活性之间的关系进行了研究。以对DNase Ⅰ消化的敏感性作为染色质的结构参数,我将β型血珠蛋白基因簇中11个区域之间以及其与不表达基因区(乳糖白蛋白和免疫球蛋白不变区λ轻链基因)的染色结构进行比较。实验的细胞系统为K 562红白血病细胞与人胚皮肤细胞株(HES)。所获得的结果提示,在细胞核内,表达基因的染色质结构疏松,对DNase Ⅰ消化的敏感性远较不表达基因区的为高。此外,本文还对有关点杂交的方法学问题进行了较为详尽的讨论。