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The integral membrane protein complex of the photosystem I reaction center from the cyanobacterium Synechococcussp. has been isolated and highly purified. At extremely low protein complex concentrations (50 μg/ml), crystallization occurred. The crystals are dichroic, photoactive and show an X-ray powder diffraction pattern.

(1) The time-course of the recombination reaction occurring after charge separation between Chl-aII(P-680) and QAin the presence of Z+was analyzed by optical spectroscopy in the wavelength region between 250 nm and 570 nm. It could be modelled by the sum of three exponential decay phases with half-lifetimes of 170 μs, 800 μs and 6 ms, where the relative amplitudes of the three phases were very similar at all wavelengths. On the basis of the absorption difference spectra it is proposed that, despite the complex time-course, only the two components Chl-aIIand QAare involved. Thereby it is supposed that the multiphasic kinetics are due to a distribution of different structural states of the reaction center proteins. (2) A difference spectrum of Chl-aII+/Chl-aIIis evaluated by subtraction of contributions due to QA−/QAfrom the combined spectrum Chl-aII+QA−/Chl-aIIQA. The latter was derived from the amplitudes of the kinetic component dominating the recombination reaction as well as from the extrapolated initial amplitudes of absorption changes measured in oxygen-evolving and in Tris-treated PS II complexes. (3) Measurements of PS II complexes incubated with acetate according to Saygin et al. (Saygin, Ö., Gerken, S., Meyer, B. and Witt, H. T. (1986) Photosynth. Res. 9, 71–78) indicated that Z is the donor which supplies electrons to Chl-aII+in this system with a half-lifetime of about 160 μs.

The number and chemical nature of the electron carrier(s) between Chl aIIand the water-oxidizing enzyme, S, were analyzed through flash-induced absorption changes in the UV with nanosecond time resolution. (i) At all wavelengths where the reaction of the donor with Chl a+IIhas been characterized, this donor is oxidized in the nanosecond time range in exact accordance with the reduction kinetics of Chl a+II. The donor is in turn re-reduced with t12> 10,μs, i.e. in the range where S is oxidized. From this time course it is concluded that there exists only one electron carrier between Chl a+IIand S. (ii) The UV-diference spectrum due to the electron transfer from the immediate donor to Chl a+IIin the nanosecond time range in O 2-evolving PS II complexes is characterized by a maximum around 260 nm and smaller minimum around 310 nm. This spectrum is identical with that observed for the reaction of the donor with Chl a+IIin the microsecond time range in Tris-treated PS II. Therefore, the donors in both reactions must be of the same chemical nature. (iii) This result, together with the well-established similarity of EPR signal II fof the oxidized donor in Tris-treated PS II to the EPR signal III s, recently assigned to Tyr-160 of the D2 protein of PS II [(1988) Proc. Natl. Acad. Sci. USA 85, 427–430], provides strong evidence that the immediate donor to Chl a+IIin water-oxidizing PS II is also a tyrosine. (iv) It is shown that the UV-difference spectra of the oxidation of the immediate donor in O 2-evolving as well as that of Tris-treated PS II complexes are similar to the in vitro difference spectrum of the oxidation of tyrosine in water. This independent result supports the conclusion that the donor is a tyrosine.

Flash‐induced absorption changes in the UV have been monitored with nanosecond time resolution in oxygen‐evolving photosystem II complexes from Synechococcussp. At 260 nm, the oxidation of the immediate donor to Chl‐a+II(P‐680+) in the 10–500 ns range could be observed directly. The difference extinction coefficient for its oxidation is estimated to be 6000–8000 M−1·cm−1at 260 nm. The observed kinetics for the oxidation of the donor is identical with the reduction kinetics of Chl‐a+IIas monitored at 824 nm. At 320 nm, the reduction of QAwas found to be faster than 1 ns.

Flash-induced absorption changes in the UV have been monitored with nanosecond time resolution in oxygen-evolving photosystem II complexes from Synechococcussp. At 260 nm, the oxidation of the immediate donor to Chl- a+II(P-680 +) in the 10–500 ns range could be observed directly. The difference extinction coefficient for its oxidation is estimated to be 6000–8000 M −1·cm −1at 260 nm. The observed kinetics for the oxidation of the donor is identical with the reduction kinetics of Chl- a+IIas monitored at 824 nm. At 320 nm, the reduction of Q Awas found to be faster than 1 ns.

Chinese hamster ovary cell lines that are 1000-fold more resistant to the threonyl-tRNA synthetase inhibitor borrelidin than the sensitive parental cells were isolated after stepwise selection for growth in increasing concentrations of the drug. These cells show a 10-20-fold increase in threonyl-tRNA synthetase activity. Quantitation of the amount of threonyl-tRNA synthetase protein by immunological techniques indicated a 60-100-fold increase compared to sensitive cells. No significant changes in the Km for substrates, inhibition by borrelidin or thermal stability were found for the threonyl-tRNA synthetase of resistant cells. These data suggest that the resistant cell lines may have amplified the gene encoding threonyl-tRNA synthetase, but no evidence of homogeneously staining regions or double minute chromosomes was found. The resistant cell lines should prove useful for the study of the regulation of threonyl-tRNA synthetase.

Oxygen evolution and reduction kinetics of the photooxidized Chl-aII+ have been measured in oxygen-evolving complexes from the thermophilic cyanobacterium Synechococcus sp.

The number and chemical nature of the electron carrier(s) between Chl aIIand the water‐oxidizing enzyme, S, were analyzed through flash‐induced absorption changes in the UV with nanosecond time resolution. (i) At all wavelengths where the reaction of the donor with Chl a+IIhas been characterized, this donor is oxidized in the nanosecond time range in exact accordance with the reduction kinetics of Chl a+II. The donor is in turn re‐reduced with t> 10,μs, i.e. in the range where S is oxidized. From this time course it is concluded that there exists only one electron carrier between Chl a+IIand S. (ii) The UV‐diference spectrum due to the electron transfer from the immediate donor to Chl a+IIin the nanosecond time range in O2‐evolving PS II complexes is characterized by a maximum around 260 nm and smaller minimum around 310 nm. This spectrum is identical with that observed for the reaction of the donor with Chl a+IIin the microsecond time range in Tris‐treated PS II. Therefore, the donors in both reactions must be of the same chemical nature. (iii) This result, together with the well‐established similarity of EPR signal IIfof the oxidized donor in Tris‐treated PS II to the EPR signal IIIs, recently assigned to Tyr‐160 of the D2 protein of PS II [(1988) Proc. Natl. Acad. Sci. USA 85, 427–430], provides strong evidence that the immediate donor to Chl a+IIin water‐oxidizing PS II is also a tyrosine. (iv) It is shown that the UV‐difference spectra of the oxidation of the immediate donor in O2‐evolving as well as that of Tris‐treated PS II complexes are similar to the in vitro difference spectrum of the oxidation of tyrosine in water. This independent result supports the conclusion that the donor is a tyrosine.

We have developed a new and simple method for quantitatively analyzing global gene expression profiles from cells or tissues. The process, called TALEST, or tandem arrayed ligation of expressed sequence tags, employs an oligonucleotide adapter containing a type IIs restriction enzyme site to facilitate the generation of short (16 bp) ESTs of fixed position in the mRNA. These ESTs are flanked by GC-clamped punctuation sequences which render them resistant to thermal denaturation, allowing their concatenation into long arrays and subsequent recognition and analysis by high-throughput DNA sequencing. A major advantage of the TALEST technique is the avoidance of PCR in all stages of the process and hence the attendant sequence-specific amplification biases that are inherent in other gene expression profiling methods such as SAGE, Differential Display, AFLP, etc. which rely on PCR.