Analysis of human metaphase chromosomes using antibodies to double-stranded and single-stranded DNA: staining patterns are related to DNA conformation

Fresh and 6-day-old fixed chromosome spreads, both untreated and treated with various banding techniques and nucleases, were stained using monoclonal antibodies to double-stranded and single-stranded DNA. DNA in fixed chromosome preparations became progressively denatured with ageing. The staining pattern of untreated chromosomes with anti-double-stranded DNA antibodies (which resembles G-banding) was determined by the conformation of the chromosomal DNA.     

Isolation and1H-NMR spectroscopic identification of the glucose-containing lipid-linked precursor oligosaccharide ofN-linked carbohydrate chains

The lipid-linked precursor ofN-type glycoprotein oligosaccharides was isolated from porcine thyroid microsomes after in cubation with UDP[³H] Glucose. The carbohydrate was released from dolichol pyrophosphate by mild acid hydrolysis, purified by gel filtration and characterized by 500-MHz¹H-NMR spectroscopy in combination with enzymatic degradation. The parent oligosaccharide was found to be Glc₃Man₉Glc-NAc₂. The three glucose residues are present in the linear sequence Glcα1-2Glα1-3 Glc, the latter being α(1-3)-linked to one of the mannose residues. In order to establish the branch location of the triglucosyl unit, the parent compound was digested with jack-bean α-mannosidase. The oligosaccharide product was purified by gel filtration, and identified by¹H-NMR as Glc₃Man₅GlcNAc₂ lacking the mannose residues A, D₂, B and D₃. Therefore, the structure of the precursor oligosaccharide is as follows: $$\begin{gathered} c b a D_1 C 4 \hfill \\ Glc\alpha 1 - 2Glc\alpha 1 - 3Glc\alpha 1 - 3Man\alpha 1 - 2Man\alpha 1 - 2Man\alpha 1 \hfill \\ 3 \swarrow 3 2 1 \hfill \\ Man\alpha 1 - 2Man\alpha 1 Man\beta 1 - 4GlcNAc\beta 1 - 4GlcNAc \hfill \\ D_{2 } A 3 6 \hfill \\ Man\alpha 1 \hfill \\ 6 \hfill \\ Man\alpha 1 - 2Man\alpha 1 \nwarrow 4 \hfill \\ D_3 B \hfill \\ \end{gathered} $$      

Effects of medium carbon-to-nitrogen ratio on biofilm formation and plasmid stability

Biofilm formation and plasmid segregational instability in biofilm cultures of Escherichia coli DH5alpha (pMJR1750) were investigated under different medium-carbon-to-nitrogen (C/N) ratios. At C/N ratios of 0.07 and 1, net accumulation of both biofilm plasmid-bearing and plasmid-free cells continued through the entire experiment without attaining any apparent steady state. At C/N ratios of 5 and 10, net biofilm cell accumulation for the two populations reached apparent steady states after 84 and 72 h, respectively. At C/N ratios of 0.07 and 1, polysaccharide production increased slowly and reached about 2g alginate equivalent/cm(2) by the end of both experiments. At a C/N ratio of 5, polysaccharide increase significantly after 84 h, reaching about 7mug alginate equivalent/cm(2) prior to termination. At a C/N ratio of 10, polysaccharide increased significantly after 72 h and reached 21 mug alginate equivalent/cm(2) at 108 h. At C/N ratios of 0.07 and 1, protein production reached 6.5 and 4 mug/cm(2), respectively. At C/N ratios of 5 and 10, protein production increased slightly for the first 84 h and reached a maximum at 108 h, at 3 and 2 mug/cm(2), respectively, then decreased over the last 12 h of the experiment. Ratios of polysaccharide to protein increased with increasing C/N ratios. At C/N ratios of 0.07 and 1, the ratios between extracellular polysaccharide (EP) and protein were no more than 205 mug polysaccharide/mug protein, whereas those at C/N ratios of 5 and 10 increased to about 7 and 12 mug polysaccharide/mug protein, respectively.Probabilities of plasmid loss in the biofilm cultures increased with increasing C/N ratios. At C/N ratios of 0.07, 1, and 5, the probabilities of plasmid loss were 0.0013 +/- 0.011, 0.020 +/- 0.006 and 0.122 +/- 0.021, respectively. At a C/N ratio of 10, the probability of plasmid loss was significantly higher, reaching 0.38 +/- 0.125. The increase of probability of plasmid loss at higher C/N ratios results from competition between cell replication and extracellular polysaccharide production. (c) 1994 John Wiley & Sons, Inc.     

Plasmid retention and gene expression in suspended and biofilm cultures of recombinant Escherichia coli DH5alpha(pMJR1750)

Differences in plasmid retention and expression are studied in both suspended and biofilm cultures of Escherichia coli DH5alpha(PMJR1750). An alternative mathematical model is proposed which allows the determination of plasmid loss probability in both suspended batch and continuously fed biofilm cultures. In our experiments, the average probability of plasmid loss of E. coli DH5alpha(pMJR1750) is 0.0022 in batch culture in the absence of antibiotic selection pressure and inducer. Under the induction of 0.17 MM IPTG, the maximum growth rate of plasmid-bearing cells in suspended batch culture dropped from 0.45 h(-1) to 0.35 h(-1) and the beta-galactosidase concentration reached an experimental maximum of 0.32. pg/cell 4 hours after the initiation of induction. At both 0.34 and 0.51 mM IPTG, growth rates in batch cultures decreased to 0.16 h(-1), about 36% of that without IPTG, and the beta-galactosidase concentration reached an experimental maximum of 0.47 pg/cell 3 hours after induction.In biofilm cultures, both plasmid-bearing and plasmid-free cells in increase with time reaching a plateau after 96 hours n the absence of both the inducer and any antibiotic selection pressure. Average probability of plasmid loss for biofilm-bound E. coli DH5beta(pMJR1750) population was 0.017 without antibiotic selection. Once the inducer IPTG was added, the concentration of plasmid-bearing cells in biofilm dropped dramatically while plasmid-free cell numbers maintained unaffected. The beta-galactosidase concentration reached a maximum in all biofilm experiments 24 hours after induction; they were 0.08, 0.1, and 0.12 pg/cel under 0.17, 0.34, and 0.51 mM IPTG, respectively. (c) 1993 John Wiley & Sons, Inc.     

Characterization of the mutant lytic state in lambda expression systems

The two propagative phases of bacteriophage lambda, lysogeny and lysis, can be used in concert to enhance productivity of recombinant expression systems. Lambda vectors carrying mutations to prevent both cell lysis and lambda DNA packaging in the lytic state have been shown to yield 100% stability of the product gene in lysogeny and to produce up to 15% of total cell protein as product beta-galactosidase in a mutant lytic state.(14) Despite these mutations, partial lysis of the culture was observed following induction of the cells from a lysogenic phase into the lytic state. To understand better the phage-host cell interactions and to investigate the possible cause(s) of lysis in these highly productive expression systems, we have made a detailed study of the suppressor-free system JM105(NM1070). We have found high levels of product (15% of total cell protein as beta-glactosidase) to be due chiefly to a high-copy number of lambda DNA in the mutant lytic state. There is partial lysis of the culture even in this suppressor-free system caused by a low-level natural suppression of the amber mutation in gene S of NM1070, resulting in accumulation of lambda endolysin. We have also monitored changes in cell growth and morphology upon induction of the lysogen. There is a slight increase in cell number that follows a linear relationship with time and a 25-fold increase in cell volume during recombinat protein production in the mutant lytic state.     

Analysis of productivity in lysis-deficient lambda expression systems

The integrated state of lambda in the host chromosome in lysogeny can be combined with its extrachromosomal replication in the lytic state to achieve high cloned gene productivities. Our previous studies on lambda expression systems(21,22) have shown 100% segregational stability of the cloned gene in lysogeny and cloned gene product levels up to 15% of total cell protein in a mutant lytic state. However, the expression phase of systems based on Escherichia coli JM109 and JM105 showed partial lysis of the productive culture despite a mutation in the lysis gene S of the lambda vector resulting in extracellular release of the cloned gene product. In the current study, we have eliminated partial lysis in the expression phase of lambda systems and conducted a detailed comparative analysis of these systems in relation to maximization of cloned gene productivity. The elimination of partial cell lysis by using a nonpermissive strain Y1089 did not enhance product yields vs. earlier systems that exhibited partial lysis. The elimination of nonessential lambda protein production by construction of a new vector NP326 did not yield higher product yields presumably because of the small fraction of these proteins in the lytic state. Temperature induction of the lysogen Y1089(NM1070) resulted in higher product levels than direct infection of Y1089 by the phage vector at a high multiplicity. Using infection experiments, we found the promoter lacUV5 in the vector lambdaZEQS to yield threefold higher product levels than lac in NM1070, suggesting possible further enhancement of productivity with stronger promoters. The occurrence or absence of partial lysis in lambda systems could be used beneficially to achieve extracellular or intracellular product as desired. The large capacity of lambda vectors for insert DNA suggests potential applications in obtaining highly amplified levels of operons and multienzyme systems. (c) 1992 John Wiley & Sons, Inc.     

Consistency of females’ stridulatory behaviour during inter‐sexual interactions in spiders

In a sexual context, it is expected that females base their choice of mate on the behaviours that males perform during courtship, as such behaviours are associated with the male's mate quality. Stridulation is one form of female communication in arthropods, for example, spiders. In spiders, stridulation during sexual interactions is relatively common in some groups but mainly restricted to males. In the pholcid spider Holocnemus pluchei (Pholcidae), both sexes have stridulatory organs. The aims of the present work were to: (a) determine possible differences in the frequency of occurrence of stridulation between females during inter‐sexual interactions, (b) establish female consistency in stridulation along repeated interactions and (c) analyse if female stridulation is associated with certain male behaviours during pre‐copulatory courtship and with male size. Female H. pluchei showed highly repeatable differences in their frequency of stridulation across consecutive encounters with males (ICC = 0.64). However, only a modest level of repeatability was detected in total time females spent stridulating across trials (ICC = 0.19). Females’ mean stridulatory behaviour did not change across ten consecutive trials spread across 20 days, and their behaviour was apparently unaffected by male persistence of copulatory attempted and/or size. These results imply that the frequency of female stridulatory behaviour is a trait that is highly characteristic of each individual. Finally, our work opens the door to determine whether behavioural consistency manifests in other ecological contexts and their functional implications.     

LIMK1 regulates Golgi dynamics, traffic of Golgi-derived vesicles, and process extension in primary cultured neurons

In this study, we examined the subcellular distribution and functions of LIMK1 in developing neurons. Confocal microscopy, subcellular fractionation, and expression of several epitope-tagged LIMK1 constructs revealed that LIMK1 is enriched in the Golgi apparatus and growth cones, with the LIM domain required for Golgi localization and the PDZ domain for its presence at neuritic tips. Overexpression of wild-type LIMK1 suppresses the formation of trans-Golgi derived tubules, and prevents cytochalasin D-induced Golgi fragmentation, whereas that of a kinase-defective mutant has the opposite effect. Transfection of wild-type LIMK1 accelerates axon formation and enhances the accumulation of Par3/Par6, insulin-like growth factor (IGF)1 receptors, and neural cell adhesion molecule (NCAM) at growth cones, while inhibiting the Golgi export of synaptophysin-containing vesicles. These effects were dependent on the Golgi localization of LIMK1, paralleled by an increase in cofilin phosphorylation and phalloidin staining in the region of the Golgi apparatus, and prevented by coexpression of constitutive active cofilin. The long-term overexpression of LIMK1 produces growth cone collapse and axon retraction, an effect that is dependent on its growth cone localization. Together, our results suggest an important role for LIMK1 in axon formation that is related with its ability to regulate Golgi dynamics, membrane traffic, and actin cytoskeletal organization.