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SUMMARY: In the light of recently revised German legislation on on-farm nitrogen (N) management, the impact of decreasing dietary N-load at balanced dietary amino acid supply on growth performance and N-excretions was examined in 5000 mixed-sex Ross 308 broilers from 1 to 40 d of age (20 floor pens with 250 birds each). In addition to diets representing the German standard with 22.0, 20.6, 20.0, and 19.5% crude protein (CP) in starter (1–10 d), grower I (11–16 d), grower II (17–30 d), and finisher feed (31–40 d), a N-reduced German standard (21.0%, 20.0%, 19.6%, and 18.9% CP) was fed in treatment 2. Dietary CP was further reduced in treatment 3 (21.0%, 19.5%, 18.7%, and 18.0% CP) and treatment 4 (21.0%, 19.0%, 18.0%, and 17.0% CP). Growth performance, feed conversion (FCR), carcass quality, N-utilization and litter quantity (at termination) were examined. Final body weight were similar between treatments 1, 2, and 3 but slightly impaired in treatment 4 (P < 0.05) while FCR was not affected. Carcass and breast meat yield did not differ significantly between treatments in male broilers. In female broilers, carcass yield was similar between treatments 1, 2, and 3 whereas it was lower in treatment 4 (P < 0.05). Breast meat yield of female broilers did not differ between treatments (P > 0.05). Dietary N-reduction resulted in gradually improved N-utilization (P < 0.05) resulting in reduction of N-excretion (P < 0.05). Moreover, quantity of litter at termination of the trial gradually decreased with dietary protein while dry matter content of litter increased and N-content decreased (P < 0.05).

An efficient low redundancy DNA sequencing strategy should allow high accuracy determination of the consensus sequence on both strands of a DNA fragment from a minimal number of sequencing reactions with minimal overlap. At EMBL we developed a directed strategy for cosmid-scale sequencing based on primer walking, whereas most other sequencing projects of this scale rely on the random ‘shotgun’ strategy. In our strategy, highly accurate raw data are obtained from automated double-stranded Sanger dideoxy sequencing with inexpensive walking primers (8 to 10 $ per primer), T7 DNA polymerase and internal labelling by fluorescein-15- ∗ dATP on A.L.F. DNA sequencers (Pharmacia Biotech). The use of 60-cm long glass plates enables reading length of up to 1000 bases. Comparing various random and directed sequencing strategies in the course of the European Community yeast genome sequencing project on cosmids from chromosomes IX, XI and XV, primer walking was found to be the strategy resulting in the lowest possible redundancy of 2.6 to 2.8. Future development of the sequencing strategy is based on the new EMBL 2-dye sequencing device for simultaneous sequencing on both strands, and implementation of an initial limited random sequencing phase to reduce the number of walking primers required by a factor of 3, while still maintaining a low redundancy of approx. 3.

A simple system has been designed enabling ultrasensitive on-line detection of fluorescently labelled macromolecules, e.g. nucleic acids, proteins and peptides during electrophoretic separations in gels. An important application is the automated DNA sequence determination without radioactivity. Drying of gels, film exposure and handling are not necessary. A sulphydryl containing M13 sequencing primer has been synthesised and end-labelled in a reaction with fluorescein iodoacetamide. This is then used in the dideoxy reactions. In particular no moving parts or complicated software are required for data collection and analysis. Compared to our first automated device detection sensitivity has been improved by a factor of thirty to about 3 X 10(-18) mol per band. The resolution has increased to about 400 bases in 5 hours, with the possibility to read up to about 500 bases when they are properly labelled. Gels shorter than 20 cm may be used for resolution of about 300 bases. The single gel system may be upgraded for simultaneous running and reading of six or ten sequencing samples.

T7 DNA polymerase with chemically inactivated 3'-5' exo-nuclease activity, as well as unmodified T7 DNA polymerase, were used for sequencing by the dideoxy method in an automated system with fluorescence labelled primer and on-line detection of laser-excited reaction products. An analysis of signal intensity variations in the C track revealed that low C signals were usually preceded by a T in the sequence. This effect was modified by surrounding nucleotides. Signal intensities were more uniform with T7 polymerase than with the Klenow fragment of DNA polymerase I. Some sequences ambiguous with the Klenow enzyme could easily be evaluated with the T7 enzyme. One sequence could only be read by the unmodified T7 polymerase, while both the Klenow fragment and the chemically modified T7 enzyme gave uninterpretable data.