Mutations affecting cyclic phosphodiesterases and adenylate cyclase in Neurospora

Summary Two regulatory mutants in orthophosphateregulated cyclic phosphodiesterase (CPDase), cpd-3 and cpd-4, were isolated and mapped proximal to arg-1 on L.G. IC and distal to urg-12 on L.G. IIR, respectively. cpd-3 showed short aerial hyphae with dense formation of conidia. The morphology was very similar to that of cr-1, cpd-3 and cr-1 had reduced levels of cyclic 3,5-AMP, adenylate cyclase and cPDases (CPDase I, II and III in low phosphate condition) but had elevated levels of cyclic 3,5-GMP. Although cr-1 showed an enhanced level and enhanced activation of heat activated cyclic phosphodiesterase (ha-PDE), this enzyme was not activated in cpd-3. cpd-4, nuc-1 and nuc-2 produced neither CPDase I, II, III, alkaline phosphatase nor ribonuclease N1 in low phosphate media. These mutants did not produce aerial hypha or conidium when grown in low phosphate liquid medium. Activation of ha-PDE occurred in cpd-4, but not in nuc-1 and nuc-2.     

Examination of a purified A′ influenza virus preparation by potentiometry

Summary The potentiometric titration of a purified influenza A virus preparation revealed 100.7×10^–4 M base and 68.8 × 10^–4 M acid-binding capacity per g. of virus protein N. The titration curve was characterized by the following fourpK values:pK ₁ = 3.37;pK ₂ = 4.50;pK ₃ = 6.37, andpK ₄ = 9.75. The isoionic point was at pH 5.43.Attempt was made to identify the dissociating groups and it was found that the carboxylic groups (pK ₁ andpK ₂) may either be glutamyl or aspartyl groups, while the cationic groups are probably the imidazolium of histidine (pK ₃) and the -amino residues of lysine (pK ₄).Inaotivation of the hemagglutinating activity of the virus preparation by mild treatment with formaldehyde at pH 8.0 resulted in a simultaneous disappearence of the -amino groups of lysine (pK ₄). The same treatment at pH 9.0 resulted in the loss of all the cationic groups previously demonstrable.The possible role of the stable positive charges on the surface of the virus at physiological pH is discussed from the point of view of the physico-chemistry of the hemagglutination.     

Komplement- und Antikörper-Titer bei Patienten mit chronischer Bronchitis

Zusammenfassung Bei insgesamt 76 Patienten mit chronischer Bronchitis wurden Komplement(C)- und Antikörper(Ak)Titer (gegen die jeweiligen aus Sputumproben gezüchteten Keime) bestimmt. Die C-Titer (CH50/ml) lagen bei 57 Patienten (= 75%) im Normbereich, bei 17 Patienten (= 22,4%) oberhalb und bei 2 Patienten (= 2,6%) unterhalb der Grenzwerte bei Kontrollpersonen. Dabei tendierten bei höheren Ak-Titern die C-Titer zu niedrigeren Werten. Es wird vermutet, daß kontinuierliche Immunreaktionen den relativen Abfall der C-Titer bei gleichzeitig hohen Ak-Titern bedingen.

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Summary In 76 patients with chronic bronchitis total complement (C) activity and antibody titer (versus correspondent microorganisms cultured from sputum samples) were determined. 57 patients (= 75%) were found to have normal C titers (CH50/ml), in 17 cases (= 22,4%) the C titers were elevated above and in 2 cases (= 2,6%) reduced under limiting values of control persons. The C titers tended to lower levels when antibody titers increased. It is supposed that continuous immune reactions cause the relative decrease of complement activity when antibody titers are increased.

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Mit finanzieller Beihilfe der Europäischen Gemeinschaft für Kohle und Stahl durchgeführte Forschungsarbeit.     

Effect of analogs of cinanserin on human lymphocytes in vitro

Cinanserin, 2-(3-dimethylaminopropylthio)cinnamanilide, was compared in phytohemagglutinin (PHA)-treated human lymphocytes in vitro to two analogs, 2-(3-dimethylaminopropoxy)-5-methylcinnamanilide (SQ 11,276) and 5-tert.-butyl-2-(3-dimethylaminopropoxy) cinnamanilide (SQ 11,332). These analogs, like cinanserin, exhibit immunosuppressive activity but, unlike cinanserin, possess low antiserotonin activity.Within 1–1.5 hours after addition of the drugs (0.15 mM), incorporation of³H-uridine,¹⁴C-leucine, and³H-thymidine into a macromolecular fraction of PHA-treated cells were inhibited, in an increasing order, by cinanserin, SQ 11,276 and SQ 11,332. At this concentration cellular recovery and viability wre unaltered by cinanserin and SQ 11,276 and substantially decreased by SQ 11,332. SQ 11,332 was less cytotoxic at lower concentrations and 4–7 times more active than cinanserin as an inhibitor of the incorporation of the nucleosides into the macromolecular fraction.     

3′-Deoxy-3′-fluoroinosine as a potent antileishmanial agent

We studied the antileishmanial activity of 3-deoxy-3-fluoroinosine (3-FI) againstLeishmania tropica andL. donovani. In in vitro cultivation, the EC₅₀ values (the concentration of drug necessary to inhibit the growth rate of cells to 50% of the control value) obtained for 3-FI against the promastigotes ofL. tropica andL. donovani were 2.3×10^–7 and 1.0×10^–6 M, respectively. It was less toxic toward mouse mammary-tumor FM3A cells, a model host; the EC₅₀ value was 1.9×10^–4 M. Leishmania promastigote metabolized 3-FI to 3-deoxy-3-fluoroadenosine 5-triphosphate (3-FATP) but FM3A cells did not. 3-FI was effective againstL. donovani amastigotes in J774.1 cells in an in vitro cultivation system under conditions similar to those used in the in vivo assay. 3-FI (50 mg/kg, given i.v.)showed a cytotoxic effect against the amastigotes ofL. donovani in mice.     

Rhinovirus detection using probes from the 5′ and 3′ end of the genome

Summary This study investigated the abilities of cDNA probes from the 5 and 3 ends of the genome of human rhinoviruses (HRV-) 14, 9, and 1B to detect RNA from 59 rhinovirus serotypes. The results show that probes from the 5 end of the genomes of HRV-14, 9, and 1B detected a large number of serotypes but the detection rate was variable and depended on the degree of homology with the particular probe. In contrast, all the 3 end probes were specific for the homologous virus. However, along HRV-9 probe detected a large number of serotypes.It was concluded that such cDNA probes would not detect all serotypes with equal efficiency. Synthetic oligonucleotides corresponding to short but highly conserved regions in the 5 non coding region may overcome this problem.     

ATP-Dependent inhibition of Ca2+-activated K+ channels in vascular smooth muscle cells by neuropeptide Y

Neuropeptide Y(NPY) inhibits Ca²⁺-activated K⁺ channels reversibly in vascular smooth muscle cells from the rat tail artery. NPY (200 M) had no effect in the absence of intracellular adenosine 5triphosphate (ATP) and when the metabolic poison cyanide-M-chlorophenyl hydrozone (10 M) was included in the intracellular pipette solution. NPY was also not effective when ATP was substituted by the non-hydrolysable ATP analogue adenosine 5-[, -methylene]-triphosphate (AMP-PCP). NPY inhibited Ca²⁺-activated K⁺ channel activity when ATP was replaced by adenosine 5-O-(3-thiotriphosphate) (ATP [-S]) and the inhibition was not readily reversed upon washing. Protein kinase inhibitor (1 M), a specific inhibitor of adenosine 3, 5-cyclic monophosphatedependent protein kinase, had no significant effect on the inhibitory action of NPY. The effect of NPY on single-channel activity was inhibited by the tyrosine kinase inhibitor genistein (10 M) but not by daidzein, an inactive analogue of genistein. These observations suggest that the inhibition by NPY of Ca²⁺-activated K⁺ channels is mediated by ATP-dependent phosphorylation. The inhibitory effect of NPY was antagonized by the tyrosine kinase inhibitor genistein.     

Effects of 5-hydroxytryptamine,dopamine, and acetylcholine on accumulation of cyclic AMP and cyclic GMP in the anterior byssus retractor muscle ofMytilus edulis L. (Mollusca)

We investigated in vitro accumulation of adenosine 3,5-monophosphate (induced by 5-hydroxytryptamine and dopamine) and of guanosine 3,5-monophosphate (induced by acetylcholine) in the anterior byssus retractor muscle ofMytilus.The response to 5-hydroxytryptamine exceeded that induced by equimolar concentrations of dopamine.1-methyl lysergic acid, a 5-hydroxytryptamine-blocking agent, diminished the 5-hydroxytryptamine-induced increase of cyclic AMP level. This parallels the effect of this amine on the contracted muscle.Acetylcholine, which causes a tonic contraction of the muscle, increased intracellular levels of cyclic GMP in a dose-dependent (max. 45-fold at 10^–4 M ACh) manner. The time course of the rise in cyclic GMP level was rapid and transient (peak concentration of cyclic GMP at 2 min).Mytolon was the most effective of all cholinergic blockers tested. It was concluded that cyclic nucleotides may play a role in the modulatory process of the transmitters. A direct relation to the relaxation-contraction process could not be established.Abbreviations ABRM anterior byssus retractor muscle - cyclic AMP adenosine 3,5-monophosphate - cyclic GMP guanosine 3,5-monophosphate - ACh acetylcholine - 5-HT 5-hydroxytryptamine, serotonin - DA dopamine - TCA trichloroacetic acid - UML 1-methyl lysergic acid hydrogen maleinate     

The trans-spliced intron 1 in the psaA gene of the Chlamydomonas chloroplast: a comparative analysis

In the secondary structure model that has been proposed for the trans-spliced intron 1 in the Chlamydomonas reinhardtii psaA gene, a third RNA species (tscA RNA) interacts with the 5 and 3 intron parts flanking the exons to reconstitute a composite structure with several features of group-II introns. To test the validity of this model, we undertook the sequencing and modelling of equivalent introns in the psaA gene from other unicellular green algae belonging to the highly diversified genus Chlamydomonas. Our comparative analysis supports the model reported for the C. reinhardtii psaA intron 1, and also indicates that the 5 end of the tscA RNA and the region downstream from the psaA exon 1 cannot be folded into a structure typical of domain I as described for most group-II introns. It is possible that a fourth RNA species, yet to be discovered, provides the parts of domain I which are apparently missing.     

Chest wall kinematics and respiratory muscle coordinated action during hypercapnia in healthy males

The present study was designed to verify whether during hypercapnic stimulation, as we had previously found during exercise or walking, the partitioning of the respiratory motor output is equally distributed to the muscles of chest wall compartments to assist diaphragm function. We studied chest wall kinematics and respiratory muscle recruitment in seven healthy men during rebreathing of a hypercapnic-hyperoxic gas mixture (CO₂ RT). Data were compared with those previously obtained during either cycling exercise or walking. The chest wall volume (Vcw), assessed by optoelectronic plethysmography (OEP), was modeled as the sum of the volumes of the lung-apposed rib cage (Vrc,p), diaphragm-apposed rib cage (Vrc,a) and abdomen (Vab). Esophageal (Pes), gastric (Pga) and transdiaphragmatic (Pdi=Pga–Pes) pressures were simultaneously recorded. Velocity of shortening (V) and power (W=PxV) of the diaphragm (Wdi), rib cage muscles (Wrcm) and abdominal muscles (Wabm) were also calculated. During CO₂ RT the progressive increase in end-inspiratory Vcw resulted from an increase in both end-inspiratory Vrc,p and Vrc,a, while the progressive decrease in end-expiratory Vcw was entirely due to the decrease in end-expiratory Vab. The increase in Vrc,p was proportionally slightly greater than that in Vrc,a. The end-inspiratory increase and end-expiratory decrease in Vcw were accounted for by inspiratory rib cage (RCM,i) and abdominal (ABM) muscle recruitment, respectively. Wdi, Wrcm and Wabm progressively increased. However, while most of Wdi was expressed in terms of velocity of shortening, most of Wrcm and Wabm was expressed as force or pressure. A comparison of CO₂ results with data obtained during exercise revealed: (1) a gradual vs. an immediate response, (2) a similar decrease in Vab,e and Pabm, (3) an apparent lack of any difference in ABM recruitment, (4) less gradual ABM relaxation, (5) no drop in Pdi but a similar Wdi change and decrease in pressure-to-velocity ratio of the diaphragm. We have found that in healthy humans: (1) the increased motor output with hypercapnia is equally distributed between RCM and ABM to minimize transdiaphragmatic pressure and (2) data on chest wall kinematics and respiratory muscle recruitment are only partly in line with those obtained during walking or cycling exercise.     

ATP and G proteins affect the runup of the Ca2+ current in bovine chromaffin cells

The Ca²⁺ current recorded by the whole-cell technique in chromaffin cells shows, before the often described rundown, a transient facilitation or runup. Initial current amplitude was 570±165 pA and then it increased by 49±23% (n=19, SD) over 2±1 min in the absence of adenosine 5-triphosphate (ATP). In the presence of ATP, this process occurred with the same magnitude but it was slowed in a dose-dependent manner, lasting 17±2 min with 2 mM ATP (n=8). Since adenosine 5-diphosphate (ADP) does not reproduce this ATP effect, a complex series of phosphorylations is likely to intervene and we show that, at least, a cAMP-dependent i.e., cyclic adenosine monophosphate) phosphorylation occurs. Pertussis toxin (PTX) pretreatment yielded an already maximal Ca²⁺ current (around 1000 pA) at the time of the patch rupture, which only slightly increased thereafter (10%, n=11). Also, guanosine 5-diphosphate (GDP) and guanosine 5-O-(2-thiodiphosphate) (GDP[s]), induced a fast runup, which was absent in the presence of GTP. Furthermore, we show that facilitation does not occur in the presence of dihydrophyridine (DHP) antagonists. Globally, our data suggest that an ATP-dependent phosphorylation stabilizes the inhibitory control exerted by a PTX-sensitive G protein and, as a result, slows down the facilitation of L-type Ca²⁺ channels. The recruitment of L-type channels can also be facilitated by the application of a DHP agonist or a depolarizing prepulse protocol. We show that these processes are only effective over a period which parallels the runup and are not additive to it. This suggests that a single process may underlie these various types of facilitation.     

A Neurospora crassa heat-shocked cell lysate translates homologous and heterologous messenger RNA efficiently,without preference for heat shock messages

Summary Cell-free protein synthesis systems were prepared from normally-grown (N-lysate) and heat-shocked (HS-lysate) Neurospora crassa mycelium. Although both lysates translated homologous mRNA, the HS-lysate was more active, yielding a higher incorporation of [³⁵S]-methionine into hot TCA-insoluble material and a vastly superior protein synthesis profile. The optimal temperature for translation by both lysates was 21 °C; the HS-lysate did not translate heat-shock mRNA preferentially at any temperature tested. Fortuitously, heterologous messenger RNAs from diverse eukaryotic and viral sources — Drosophila, dog pancreas, rabbit globin mRNA, brome mosaic virus, tobacco mosaic virus — were translated by the HS-lysate with an efficiency comparable to that of the commercial rabbit reticulocyte system and superior to the wheat germ system. The cap analogues, m⁷G(5)ppp(5)G and m⁷G(5)ppp(5)Gm, inhibited translation significantly.     

Phosphorylation of caldesmon by smooth-muscle casein kinase II

Summary A caldesmon kinase activity was partially purified from an extract of chicken gizzard smooth muscle by sequential chromatography on columns of DEAE-Sephacel, MonoQ and Superose 12. This kinase was identified as casein kinase II by Western blotting using peptide-directed antibodies raised against the , and subunits of human casein kinase II; the smooth muscle enzyme consisted of similar subunits of M_r 43 000 (), 39 000 (), and 27 000 (). Phosphorylation of caldesmon and casein by smooth muscle casein kinase II was optimal at 0.1 M NaCl, did not require second messengers, and was inhibited by heparin. The kinase utilized either GTP or ATP as a substrate. Caldesmon was phosphorylated to 1 mol P_i mol^-1 caldesmon by smooth muscle casein kinase II with a K_m for caldesmon of 4.9 M. Two-dimensional thin-layer electrophoresis indicated phosphate incorporation into both serine and threonine. All the incorporated phosphate was recovered in the N-terminal peptide (residues 1–152) generated by cleavage at cysteine 153 with 2-nitro-5-thiocyanobenzoic acid. Purification of tryptic phosphopeptides and N-terminal sequencing revealed two principal sites of phosphorylation: serine 73 and threonine 83. The following four synthetic peptides corresponding to this domain of caldesmon were examined as substrates of casein kinase II: A = RRREVNAQNSVAEEE; B = AQNSVAEEE; C = RSTDDEAA; D = SVAEEETKRSTDDE. Interestingly, only peptides C and D were phosphorylated and both only at threonine. Phosphorylation of intact caldesmon did not affect the pattern of chymotryptic digestion suggesting that it does not induce a significant conformational change in the protein substrate. Phosphorylation also had no effect on the binding of caldesmon to actin or on the caldesmon-mediated inhibition of actomyosin MgATPase activity. However, phosphorylation completely abolished the interaction of caldesmon with immobilized smooth muscle myosin. These results are consistent with the localization of the myosin-binding domain near the N-terminus of caldesmon and of the actin-binding domain near the opposite end of the elongated molecule. Casein kinase II may therefore play a role in regulating caldesmon-myosin interaction and the ability of caldesmon to cross-link actin and myosin filaments in smooth muscle.     

Nucleotide sequence and determination of the extremities of the 26S ribosomal RNA gene in wheat mitochondria: evidence for sequence rearrangements in the ribosomal genes of higher plants

Summary The nucleotide sequence of the wheat mitochondrial 26S ribosomal RNA gene and flanking regions was determined and compared with mitochondrial 26S rRNA genes from maize and Oenothera. All three genes exhibit a high degree of homology except within two variable regions. When the plant mitochondrial 26S rRNA genes are compared with Escherichia coli 23S rRNA and chloroplast 23S and 4.5S rRNA genes, a third variable region is apparent close to the 3 end of the gene. The 5 and 3 ends of the wheat mitochondrial gene were determined by S1 nuclease mapping. Computer analysis of the wheat mitochondrial gene revealed several small sequences present either in the 5 region of the 26S rRNA gene or in the 18S rRNA gene.     

The oligodendroglial reaction to brain stab wounds: An immunohistochemical study

Summary Myelin/oligodendrocyte specific protein was compared to glial fibrillary acidic protein and 23-cyclic nucleotide 3-phosphodiesterase expression in normal rat brains and following stab wounds to the cerebral cortex, corpus callosum and hippocampus. Animals with stab wounds were allowed to recover for 5,15,28, 45 and 70 days post-operation before fixation by perfusion. Sections were reacted with antibodies against myelin/oligodendrocyte specific protein, glial fibrillary acidic protein and 23-cyclic nucleotide 3-phosphodiesterase, and observed by light and electron microscopy. Normal cerebral cortex had very few myelin/oligodendrocyte specific protein-positive and 23-cyclic nucleotide 3-phosphodiesterasepositive cells, but some glial fibrillary acidic protein-positive cells. The myelinated fibres of the corpus callosum were heavily stained for myelin/oligodendrocyte specific protein but unstained by glial fibrillary acidic protein or 23-cyclic nucleotide 3-phosphodiesterase antibodies. Some immunopositive cells were present in the corpus callosum and hippocampus with all three antibodies. After stab wound myelin/oligodendrocyte specific protein-positive reactive cells had more and longer processes and stained more intensely than equivalent cells in normal brain. These cells were distributed along the wound track, including within the cerebral cortex. The numbers of these cells increased until 28 days post-operation and then decreased so that very few were found at 70 days post-operation except in the corpus callosum. Where demyelination occurred myelin/oligodendrocyte specific protein-staining was lost. Staining for 23-cyclic nucleotide 3-phosphodiesterase revealed a similar pattern. Glial fibrillary acidic protein-positive reactive cells, which were also more robust than the normal cells, were more widely distributed. They increased in number throughout the time periods studied and gliosis was evident on the contralateral side. The glial fibrillary acidic protein-positive astrocytes were also different from the myelin/oligodendrocyte specific protein-positive and 23-cyclic nucleotide 3-phosphodiesterase-positive oligodendrocytes in terms of cell shape. With electron microscopy myelin/oligodendrocyte specific protein-positive cells showed features typical of immature oligodendrocytes. We conclude that the injury caused a numerical increase in oligodendrocytes and that myelin/ oligodendrocyte specific protein is a good marker for the oligodendroglial response and demyelination in pathological conditions.     

Studien über den Mechanismus der Immunhämolyse: Die Bindung der zweiten Komplementkomponente

Zusammenfassung Es wird die Reaktion EAC_1,4 + C₂ isoliert untersucht. Hierzu bietet man einer Charge von stufenweise aufgebautem EAC_1,4 ein von allen übrigen Komplementkomponenten befreites C₂-Präparat an. Bei der Reaktion kommt es zu einem meßbaren Schwund von C₂ im Überstand; gleichzeitig erwerben die Zellen die Fähigkeit, mit cheliertem Komplement zu lysieren. Die Reaktionsgeschwindigkeit ist bei 37° C hoch und bei 0° C gering. Für die Fähigkeit der Zellen, C₂ zu binden, ist nicht allein das gebundene C₄ maßgebend, sondern ein von C₁ nicht abgrenzbarer Zusatzfaktor. Demnach kommen dem C₁ nach seiner Bindung zwei Funktionen zu: Einmal vermittelt es die Bindung von C₄, zum anderen vermittelt es zusammen mit dem gebundenen C₄ die Bindung von C₂.Mit Unterstützung der Deutschen Forschungsgemeinschaft sowie der Gesellschaft der Freunde und Förderer der Medizinischen Akademie Düsseldorf.     

Bleomycin-kanamycin resistance as a marker of the presence of transposon Tn5 in clinical strains ofEscherichia coli

The aminoglycoside modifying enzyme aminoglycoside 3- phosphotransferase II (APH(3)II) is encoded for on transposon Tn5 by theaphA gene, in the same operon as theble gene determining bleomycin resistance. To document this linkage 82 kanamycin-resistantEscherichia coli strains of clinical origin were studied; all 18 isolates presenting bleomycin-kanamycin resistance were shown by an enzymatic assay to produce APH(3)II, and the presence of Tn5 was demonstrated by gene hybridization. Similarly, bleomycin-kanamycin resistance was shown to be linked to APH(3)II production inSalmonella spp. The epidemiology of strains with Tn5-encoded APH(3)II may thus be studied, at least inEscherichia coli, by a simple diffusion test using bleomycin and kanamycin discs.     

Characterization of the 3′-terminal nucleotide sequence of two Korean isolates of Daphne virus S support its placement as a distinct species of the genus <Emphasis Type="Italic">Carlavirus</Emphasis>

Summary. This study determined the 3-terminal nucleotide sequences of two Korean isolates of Daphne virus S (DVS), a tentative member of the genus Carlavirus, causing leaf distortion and chlorotic spot disease symptoms in daphne plants. The 3-terminal 1,465 nucleotide sequences of the two isolates contained two open reading frames coding for proteins of 36kDa viral coat protein (CP) and 12kDa from the 5–3 end, which is a typical genome structure of the 3-terminal region of carlaviruses. Both DVS isolates were 98.1% and 93.6% amino acid identical in the CP and 12kDa, respectively. The CP gene of DVS shares 25.2–55.2% and 42.9–56.1% similarities with that of 19 other carlaviruses at the amino acid and nucleotide levels, respectively. The 3-proximal 12kDa gene of DVS shares 20.2–57.8% amino acid identities with that of 18 other members of the genus. The 3 noncoding region of DVS consists of 73 nucleotides with long excluding poly A tract, and shares 69.1–77.1% identities to the known carlaviruses. In the phylogenetic analyses of the two proteins, DVS was closely related to Helenium virus S and Chrysanthemum virus B. This is the first sequence information for the DVS, and further confirms the classification of DVS as a distinct member of the genus Carlavirus.Received January 15, 2003; accepted May 21, 2003     

Regulatory sequences clustered at the 5′ end of the first intron of the human thymidylate synthase gene function in cooperation with the promoter region

A human thymidylate synthase (TS) minigene containing 5- and 3-flanking sequences, all the exons, and only intron 1 showed a normal frequency of stable transformation when transfected into TS-negative mutant cells, whereas minigenes in which intron 1 was replaced by intron 2 or deleted in the above construct showed only a few percent of the above frequency. Introduction of intron 1 into the above intronless or intron 2 minigene restored the transforming activities regardless of its position and orientation. Deletion analysis revealed two positive and one negative regulatory sequences in the 5 end of intron 1, each of which seemed to bind specific proteins as shown by gel shift analysis. Intron 1 also stimulated expression of a TS promoter-CAT gene construct but not that of an SV40 promoter-CAT gene construct. These results indicate that the multiple regulatory sequences clustered in intron 1 stimulate TS gene expression in concert with the 5-flanking sequences.