Expression of endogenous and microinjected hsp 30 genes in early Xenopus laevis embryos

In the present study, we have examined the regulation of expression of a newly isolated member of the hsp 30 gene family, hsp 30C. Using RT-PCR, we found that this gene was first heat-inducible at the tailbud stage of development. We also examined the expression of two microinjected modified hsp 30C gene constructs in Xenopus embryos. One of the constructs had 404 bp of hsp 30C 5′-flanking region, whereas the other had 3.6 kb. Both gene constructs had 1 kb of 3′-flanking region. RT-PCR assays were employed to detect the expression of these microinjected genes. The presence of extensive 5′- and 3′-flanking regions of the hsp 30C gene did not confer proper developmental regulation, since heat-inducible expression of both of the microinjected constructs was detectable at the midblastula stage. The premature expression of the microinjected hsp 30 gene was not a result of high plasmid copy number or the presence of plasmid DNA sequences. These results suggest that the microinjected genes contain all the cis-acting DNA sequences required for correct heat-inducible regulation but do not contain the elements required for the proper regulation of hsp 30 gene expression during development. It is possible that regulatory elements controlling the developmental expression of the hsp30 genes may reside upstream or downstream of the entire cluster. © 1993Wiley-Liss, Inc.     

Cytoplasm Rescues an Arrhythmic Mutant on the Circadian Rhythm of Mating Reactivity in Paramecium bursaria

Cells of an unusual Paramecium bursaria stock (Sj2) expressed rhythmic mating reactivity in a light/dark cycle (LD) and under continuous illumination (LL). When placed in continuous darkness (DD), did not show rhythmicity but rather demonstrated a continuous high mating reactivity. However, mating reactivity was reduced following exposure to a 6-h light pulse interrupting the DD, and then recovered to its former condition. Genetic analysis showed the arrhythmicity in DD to be a dominant character inherited in a Mendelian ratio. On the other hand, a clone (MCIw) that did not show the rhythmicity in either DD or LL was isolated from the parent stock Sj2w following a 5-h treatment with 2 μg/ml nitrosoguanidine (MNNG). The MCIw cells expressed weak rhythmicity in LD, but were insensitive to a 6-h light pulse in DD. The arrhythmicity in LL was inherited cytoplasmically. In addition to this, rhythmicity in LL could be recovered by injection of cytoplasm from the wild-type cell when the recipient cell was homozygous for the wild-type nuclear gene (+/+). The cytoplasmic components or factors are assumed to control the functional circadian system and genetically determine the rhythmicity of mating reactivity.     

阿片系统介导的去甲肾上腺素在兔孤束核联合亚核引起的心血管效应

于杂种白兔的孤束核联合亚核(CNTS)内微量注射去甲肾上腺素(NE),引起血压降低和心率减慢,将小剂量阿片受体阻断剂纳洛酮注入CNTS,本身不影响血压与心率,但减弱NE在CNTS中的降压降率幅度。于CNTS内微量注射盐酸吗啡,降低血压和心率;小剂量NE注入CNTS, 本身对血压和心率无明显影响,但可显著增强盐酸吗啡在CNTS中的心血管作用。结果表明,去甲肾上腺素在CNTS中的心血管效应至少部分由降压性阿片系统介导。     

DNA-mediated genetic transformation of mouse embryos and bone marrow--a review

In recent years, new gene transfer systems have been developed which allow molecularly cloned genetic material to be introduced into whole organisms. These systems include the microinjection of DNA into mammalian embryos, transfection of DNA into mouse bone marrow cells, and the infection of early embryos with retroviruses. Exogenous DNA appears to integrate randomly into the host genome. The production of transgenic mice by injection of DNA into mouse embryos has rapidly gained importance as an experimental tool for the study of gene regulation during development. Through this technique, recombinant molecules of any type can be introduced into one-celled embryos, and thus can be used to study development from its earliest stages. DNA sequences have been shown to integrate and transmit through the germ line to subsequent generations as mendelian traits. Transgenic mice carrying various gene constructs have been successfully exploited for the elucidation of factors which determine tissue specificity of gene expression as well as the level of gene control. Phenotypic changes related to expression of foreign genes have also been observed. This experimental approach thus promises to rapidly solve many of the heretofore most challenging problems in developmental genetics. Insertion of foreign genes has also made possible the creation of insertional mutants which manifest themselves most frequently as recessives. Such mutations can be readily studied at the molecular level by using the transferred material as a probe for recovery of the affected host sequence from genomic libraries. Many of these same problems have been addressed by introducing retroviral DNA into mouse embryos. Here, the sequences used for transfer have been limited to retroviral genes, but nonetheless these experiments have been profitably exploited for studies both of gene regulation and mutagenesis. Gene transfer systems are being developed allowing the experimenter to transfer DNA into bone marrow cells of mice, after which the recipient cells can be reintroduced into lethally irradiated histocompatible animals. This system has the advantage that selection can be applied during the gene transfer process such that the expression of the foreign material is assured. In addition, these experiments have created a model system for production of animals carrying a subpopulation of cells which is highly resistant to a toxic agent. This system has the potential for therapeutic application to man.     

Carboxy-terminal domain of Cas differentially modulates c-Jun expression,DNA synthesis,and membrane ruffling induced by insulin,EGF, and IGF-1

p130 Crk-associated substrate (Cas) is an adaptor protein associating with many other signaling proteins and regulates a various biological processes including cell adhesion, migration, and growth factor stimulation. However, the exact functional role of Cas in growth factor signaling pathway was poorly understood. Here we investigated the role of Cas and its domains in the effects of insulin, EGF, and IGF-1 on c-Jun gene expression, DNA synthesis, cytoskeletal reorganization. We found that microinjection of anti-Cas antibody and C-terminal domain of Cas (Cas-CT) specifically inhibited EGF-induced, but not insulin- or IGF-1-induced, c-Jun expression. Cell cycle progression and cytoskeleton reorganization induced by insulin and EGF, but not by IGF-1, were inhibited by microinjected anti-Cas and Cas-CT. In contrast, microinjection of the substate domain (Cas-SD) of Cas did not have any inhibitory effects. These results revealed that the Cas-CT is differentially implicated in insulin and EGF-mediated, but not IGF-1-mediated, c-Jun expression, DNA synthesis and membrane ruffling.     

Symplasmic connections between sieve element and companion cell in the stem phloem ofVicia faba L. have a molecular exclusion limit of at least 10 kDa

High-molecular-weight fluorochromes were intracellularly injected into a sieve element of the fascicular stem phloem ofVicia faba L., using a modified membrane-potential-recording pressure probe. After stabilization of the membrane potential following microelectrode impalement, either LYCH (Lucifer Yellow CH), 4.4-kDa FITC-dextran (fluoresceinisothiocyanate-dextran) conjugate, or 3-kDa, 10-kDa or 40-kDa LYCH-dextran conjugate was microinjected into the sieve element. Longitudinal fluorochrome movement across the sieve plates and lateral displacement to the companion cells was detected with all the probes except the 40-kDa conjugate. This indicates that the molecular exclusion limit of the pore/plasmodesma units between a sieve element and a companion cell in the fascicular stem phloem ofVicia faba lies between 10 kDa and 40 kDa.Abbreviations FITC fluoresceinisothiocyanate - LYCH Lucifer Yellow CH - MEL molecular exclusion limit - PPU pore/plasmodesma unit - SE/CC-complex sieve element/companion cell complex     

The nuclear localization sequence of the SV40 T antigen promotes transgene uptake and expression in zebrafish embryo nuclei

We report luciferase expression in zebrafish embryos after cytoplasmic injection of low copy numbers of plasmid DNA coupled to the SV40 T antigen nuclear localization sequence (NLS). Binding of NLS to plasmid DNA (pCMVL) occurs at room temperature in 0.25m KCl, as assayed by gel retardation at molar ratios of NLS:pCMVL of at least 100:1. Luciferase expression is induced in 35% of embryos with as low as 10³ NLS-bound pCMVL copies. With 10⁴ copies, the proportion of expression increases from 6% at 0:1 to 70% 100:1 NLS:pCMVL (p<0.01). The beneficial effect of NLS is abolished at DNA concentrations promoting high frequencies of transgene expression without NLS. Regardless of the DNA concentration, the use of NLS does not affect embryo viability for at least up to 10 days: The specificity of NLS on luciferase expression was tested by using a nuclear import deficient reverse NLS peptide (revNLS). revNLS binds to pCMVL, causing gel retardation similarly to NLS, but does not promote transgene expression. Binding of equimolar amounts of revNLS and NLS to DNA reduces by 50% the beneficial effect of NLS on transgene expression. The results suggest efficient targeting of NLS-bound plasmid DNA to the nucleus, and subsequent enhanced uptake of DNA by the nucleus. The data suggest that the use of NLS may reduce the need for using elevated DNA copy numbers in some gene transfer applications.     

神经激肽A注入大鼠外侧网状核或中缝大核产生的镇痛效应

本工作采用核团内微量注射的方法,以甩尾反射潜伏期（ＴＦＬ）为痛阈指标,在浅麻状态下的大鼠上,对神经激肽Ａ（ＮＫＡ）在外侧网状核（ＬＲＮ）和中缝大核（ＮＲＭ）中的作用作了探讨。研究结果表明,ＮＫＡ（０．５μｇ／０．５μｌ）注入ＬＲＮ后的１０ｍｉｎ内大鼠ＴＦＬ明显延长（ｎ＝１２,Ｐ＜０．００１）,同样剂量ＮＫＡ注入ＮＲＭ后的５ｍｉｎ内,ＴＦＬ也明显延长（ｎ＝１３,Ｐ＜０．００１）,提示ＮＫＡ可能是ＬＲＮ和ＮＲＭ内参与痛觉的一种神经调质。     

Transgenic rapeseed plants obtained by the microinjection of DNA into microspore-derived embryoids

Summary A novel method in the field of genetic engineering of higher plants is presented: microinjection into multicellular structures which have a high competence for plant regeneration through embryogenesis. Microspore-derived embryoids of Brassica napus L. were individually selected and microinjected with NPT II gene constructions. High frequency regeneration of haploid plants through embryogenesis was achieved within 8 weeks. Transformation efficiencies between 27% and 51% were determined by DNA dot blot analysis of primary regenerants. Stable integration of fulllength microinjected genes into high molecular weight DNA was proven by Southern analysis of genomic DNA isolated from regenerated plants. Transformed plants were tested for expression of the NPT II gene by enzyme assay. The chimeric nature of the primary regenerants was demonstrated after their in vitro segregation through secondary embryogenesis into pure transformants.